Infectious cDNA of North American Porcine Reproductive and Respiratory Syndrome (PRRS) Virus and Uses Thereof

ABSTRACT

The invention provides isolated polynucleotide molecules that comprise a DNA sequence encoding an infectious RNA sequence encoding a genetically-modified North American PRRS virus, wherein the polynucleotide molecule lacks at least one detectable antigenic epitope of North American PRRS virus. The invention also provides vaccines comprising genetically modified North American PRRS virus, RNA molecules, plasmids and viral vectors comprising the isolated polynucleotide molecules. Also provided are isolated polynucleotide molecules further comprising at least one nucleotide sequence that encodes a detectable heterologous antigenic epitope, and vaccines comprising North American PRRS virus, RNA molecules, plasmids and viral vectors comprising such isolated polynucleotide molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 11/366,851, filed on Mar. 1, 2006, which is acontinuation-in-part of U.S. patent application Ser. No. 10/241,332,filed Sep. 11, 2002, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/127,391, filed on Apr. 22, 2002, which is acontinuation of U.S. patent application Ser. No. 09/470,661, filed Dec.22, 1999, U.S. Pat. No. 6,500,662, which claims the benefit of priorityof U.S. Provisional Application Ser. No. 60/113,345, filed Dec. 22,1998, the disclosures of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention is in the field of animal health and is directedto infectious cDNA clones of positive polarity RNA viruses and theconstruction of vaccines, in particular, swine vaccines, using such cDNAclones.

BACKGROUND OF THE INVENTION

Porcine reproductive and respiratory syndrome (PRRS) is a new disease ofswine, first described in 1987 in North America and in 1990 in Europe.The disease has since spread to Asia and affects most of the major swineproducing countries of the world. Primary symptoms are reproductiveproblems in sows and gilts, including late term abortions, stillbirthsand mummies, and litters of small weak pigs which are born viremic andoften fail to survive. In addition, the syndrome manifests itself as arespiratory disease in young pigs which spreads horizontally and causesfever, lethargy, labored breathing, loss of appetite, slow growth, andoccasionally death, often in association with other respiratorypathogens. The disease furthermore can be transmitted to sows and giltsvia the semen of infected boars, either naturally or by artificialinsemination. For these, and other reasons, PRRS has proven to be adifficult disease to control and therefore one of the most economicallydamaging diseases to the swine industry.

The causative agent of PRRS is the PRRS virus, which exists as twogenetically and serologically distinct types (Murtaugh, M. P. et al.,1995, Arch-Virol. 140, 1451-1460; Suarez, P. et al., 1996, VirusResearch 42:159-165). The two types are believed to have first enteredswine populations independently, one in North America and the other inEurope, in the 1980's, from unknown biological reservoirs, possibly ofrodent or avian origin. The European type, represented by the prototype“Lelystad Virus”, was isolated and sequenced in the Netherlands in 1991(Terpstra, C. et al., 1991, Vet. Quart. 13:131-136; Wensvoort, G. etal., 1991, Vet. Quart. 13:121-130; Wensvoort, G. et al., WO 92/213751992(PCT/NL92/00096), 1992; Meulenberg, J. J. M. et al., 1993, Virol.192:62-72).

Both the North American PRRS virus and the European PRRS virus areclassified within the family Arteriviridae, which also includes equinearteritis virus, lactate dehydrogenase-elevating virus, and simianhaemorrhagic fever virus. The arteriviruses are in turn placed withinthe order Nidovirales, which also includes the coronaviruses andtoroviruses. The nidoviruses are enveloped viruses having genomesconsisting of a single strand of positive polarity RNA. The genomic RNAof a positive-stranded RNA virus fulfills the dual role in both storageand expression of genetic information. No DNA is involved in replicationor transcription in nidoviruses. The reproduction of nidoviral genomicRNA is thus a combined process of genome replication and mRNAtranscription. Moreover, some proteins are translated directly from thegenomic RNA of nidoviruses. The molecular biology of the familyArteriviridae has recently been reviewed by Snijder and Meulenberg(Snijder, E. J. and Meulenberg, J. J. M., 1998, Journal of GeneralVirology 79:961-979).

Currently available commercial vaccines against PRRS are eitherconventional modified live virus (cell culture, attenuated) orconventional killed (inactivated cell culture preparations of virulentvirus). Several of these vaccines have been criticized based on safetyand/or efficacy concerns. The development of a second generation of PRRSvaccines, based upon specific additions, deletions, and othermodifications to the PRRS genome, is therefore highly desirable.However, since the PRRS viruses do not include any DNA intermediatesduring their replication, such vaccines have thus far awaited theconstruction of full-length cDNA clones of PRRS viruses for manipulationby molecular biology techniques at the DNA level. Very recently, afull-length infectious cDNA clone of the European PRRS virus has beenreported (Meulenberg, J. J. M. et al., 1998, supra; Meulenberg, J. J. M.et al., 1988, J. Virol. 72, 380-387.).

The preceding publications, as well as all other references discussedbelow in this application, are hereby incorporated by reference in theirentireties.

SUMMARY OF THE INVENTION

The subject invention provides an isolated polynucleotide moleculecomprising a DNA sequence encoding an infectious RNA molecule encoding aNorth American PRRS virus that is genetically modified such that when itinfects a porcine animal it is unable to produce PRRS in the animal yetable to elicit an effective immunoprotective response against a PRRSvirus in the porcine animal, wherein said DNA sequence is SEQ ID NO:1 ora sequence homologous thereto, except that it lacks at least one DNAsequence encoding a detectable antigenic epitope of North American PRRSvirus.

The subject invention further provides an isolated polynucleotidemolecule where at least one DNA sequence encoding a detectable antigenicepitope of North American PRRS virus in ORF 1a or ORF 1b within said DNAsequence is deleted.

The subject invention further provides an isolated polynucleotidemolecule where at least one DNA sequence encoding a detectable antigenicepitope of North American PRRS virus in the nonstructural protein 2coding region of ORF1a within said DNA sequence is deleted.

The subject invention further provides an isolated polynucleotidemolecule where at least one DNA sequence encoding a detectable antigenicepitope of North American PRRS virus in the hypervariable region in thenonstructural protein 2 coding region of ORF1a within said DNA sequenceis deleted.

The subject invention further provides an isolated polynucleotidemolecule where the DNA sequence encoding amino acids 628 and 759 in thehypervariable region in the nonstructural protein 2 coding region ofORF1a within said DNA sequence is deleted.

The subject invention further provides a vaccine for protecting aporcine animal from infection by a PRRS virus, wherein said vaccinecomprises a genetically modified North American PRRS virus encoded by aninfectious RNA molecule encoded by the isolated polynucleotide moleculeas described above; an infectious RNA molecule encoded by the isolatedpolynucleotide molecule as described above; an isolated polynucleotidemolecule as described above in the form of a plasmid, or a viral vectorcomprising the isolated polynucleotide molecule as described above, anda carrier acceptable for veterinary use.

The subject invention also provides an isolated polynucleotide moleculecomprising a DNA sequence encoding an infectious RNA molecule encoding aNorth American PRRS virus that is genetically modified such that when itinfects a porcine animal it is unable to produce PRRS in the animal yetable to elicit an effective immunoprotective response against a PRRSvirus in the porcine animal, wherein the DNA sequence is SEQ ID NO:1 ora sequence homologous thereto, except that it lacks at least one DNAsequence encoding a detectable antigenic epitope of North American PRRSvirus, wherein it further comprises at least one nucleotide sequencethat encodes a detectable heterologous antigenic epitope.

The present invention further provides an isolated polynucleotidemolecule comprising at least one nucleotide sequence encoding aheterologous antigenic epitope, wherein said nucleotide sequenceencoding a heterologous antigenic epitope is inserted in ORF 1a or ORF1b within said DNA sequence encoding an infectious RNA molecule encodinga North American PRRS virus.

The present invention further provides an isolated polynucleotidemolecule comprising at least one nucleotide sequence encoding aheterologous antigenic epitope, wherein said nucleotide sequenceencoding a heterologous antigenic epitope is inserted in thenonstructural protein 2 coding region of ORF 1a within said DNA sequenceencoding an infectious RNA molecule encoding a North American PRRSvirus.

The present invention further provides an isolated polynucleotidemolecule comprising at least one nucleotide sequence encoding aheterologous antigenic epitope, wherein said nucleotide sequenceencoding a heterologous antigenic epitope is inserted in thehypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence encoding an infectious RNA molecule encodinga North American PRRS virus.

The present invention further provides an isolated polynucleotidemolecule comprising at least one nucleotide sequence encoding aheterologous antigenic epitope, wherein said nucleotide sequenceencoding a heterologous antigenic epitope is inserted in thehypervariable region in the nonstructural protein 2 coding region of ORF1a between the DNA sequence encoding amino acids 628 and 759 in thehypervariable region in the nonstructural protein 2 coding region ofORF1a within said DNA sequence.

The present invention further provides a vaccine for protecting aporcine animal from infection by a PRRS virus, wherein said vaccinecomprises a genetically modified North American PRRS virus encoded by aninfectious RNA molecule encoded by an isolated polynucleotide moleculeas described above, an infectious RNA molecule encoded by an isolatedpolynucleotide molecule as described above, an isolated polynucleotidemolecule as described above, or a viral vector comprising an isolatedpolynucleotide molecule as described above, and a carrier acceptable forveterinary use.

The present invention also provides a diagnostic kit for differentiatingbetween porcine animals vaccinated with the above described vaccines andporcine animals infected with field strains of PRRS virus. Said kitcontains as one of its components at least one peptide having a sequencecomprising the 132 amino acid region deleted within the PRRS virusstrains presently described. Alternative, the kit can have as one of itscomponents the corresponding peptide from any PRRS virus strain. The kitcan otherwise have as one of its components a smaller peptidecorresponding to a portion of the 132 amino acid sequence comprising thensp2 deletion, from 10 amino acid residues or upward in length. Thepeptide can additionally be in the form of a fusion protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Cloning strategy for construction of full-length infectious cDNAclone of North American PRRS virus, pT7P129A. Arrowheads represent T7promoter sequences.

FIG. 2: Serum viremia following infection with P129A or recombinant PRRSvirus rP129A-1. Determined by plaque assay on MARC-145 cells. The lowerlimit of detection is 5 pfu/ml (or 0.7 on the log scale).

FIG. 3: Anti-PRRS virus serum antibody following infection with P129A orrecombinant PRRS virus rP129A-1. Determined by HerdChek PRRS ELISA assay(IDEXX (Westbrook, Me., USA)).

FIG. 4: Genome organization of PRRSV virus. Location or range of variousdeletions and insertion sites, including: ORF 1a and 1b, Nsp2, thehypervariable region of Nsp2, the deleted region within Nsp2 encodingamino acids 628-759, and the MluI and SgrA1 sites at genome positions3219 and 3614, between which GFP or the 9 amino acid HA tag wereinserted. The active site residues of the cysteine protease domain(Cys437 and His507) and the hypervariable region are indicated. Thehypervariable region of nsp2 corresponds approximately to genomecoordinates 2720-3980 in P129.

DETAILED DESCRIPTION OF THE INVENTION

Production and manipulation of the isolated polynucleotide moleculesdescribed herein are within the skill in the art and can be carried outaccording to recombinant techniques described, among other places, inManiatis, et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel, etal., 1989, Current Protocols In Molecular Biology, Greene PublishingAssociates & Wiley Interscience, NY; Sambrook, et al., 1989, MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Innis et al. (eds), 1995, PCRStrategies, Academic Press, Inc., San Diego; and Erlich (ed), 1992, PCRTechnology, Oxford University Press, New York, all of which areincorporated herein by reference.

A. Isolated Polynucleotide Molecules and RNA Molecules EncodingGenetically Modified North American PRRS Viruses.

The present invention provides isolated polynucleotide moleculescomprising DNA sequences that encode infectious RNA molecules thatencode genetically modified North American PRRS viruses.

The subject invention provides an isolated polynucleotide moleculecomprising a DNA sequence encoding an infectious RNA molecule thatencodes a North American PRRS virus, wherein said DNA sequence is SEQ IDNO:1 or a sequence homologous thereto. The present invention provides anisolated polynucleotide molecule comprising a DNA sequence encoding aninfectious RNA molecule that encodes a North American PRRS virus,wherein said DNA sequence is the sequence beginning with and includingnucleotide 1 through and including nucleotide 15,416 of SEQ ID NO:1,except that the nucleotide corresponding to nucleotide 12,622 of SEQ IDNO:1 is a guanine instead of an adenine and the nucleotide correspondingto nucleotide 1,559 of SEQ ID NO:1 is a thymine instead of a cytosine.Said DNA sequence encodes an infectious RNA molecule that is the RNAgenome of the North American PRRS isolate P129.

It is understood that terms herein referring to nucleic acid moleculessuch as “isolated polynucleotide molecule”, “nucleotide sequence”, “openreading frame (ORF)”, and the like, unless otherwise specified, includeboth DNA and RNA molecules and include both single-stranded anddouble-stranded molecules. Also, when reference to a particular sequencefrom the “Sequence Listing” section of the subject application is made,it is intended, unless otherwise specified, to refer to both the DNA ofthe “Sequence Listing”, as well as RNA corresponding to the DNAsequence, and includes sequences complementary to the DNA and RNAsequences. In such contexts in this application, “corresponding to”refers to sequences of DNA and RNA that are identical to one another butfor the fact that the RNA sequence contains uracil in place of thymineand the backbone of the RNA molecule contains ribose instead ofdeoxyribose.

For example, SEQ ID NO:1 is a DNA sequence corresponding to the RNAgenome of a North American PRRS virus. Thus, a DNA sequencecomplementary to the DNA sequence set forth in SEQ ID NO:1 is a templatefor, i.e. is complementary to or “encodes”, the RNA genome of the NorthAmerican PRRS virus (i.e., RNA that encodes the North American PRRSvirus). Nonetheless, a reference herein to SEQ ID NO:1 includes both theRNA sequence corresponding to SEQ ID NO:1 and a DNA sequencecomplementary to SEQ ID NO:1.

Furthermore, when reference is made herein to sequences homologous to asequence in the Sequence Listing, it is to be understood that sequenceshomologous to a sequence corresponding to the sequence in the SequenceListing and sequences homologous to a sequence complementary to thesequence in the Sequence Listing are also included.

An “infectious RNA molecule”, for purposes of the present invention, isan RNA molecule that encodes the necessary elements for viralreplication, transcription, and translation into a functional virion ina suitable host cell, provided, if necessary, with a peptide or peptidesthat compensate for any genetic modifications, e.g. sequence deletions,in the RNA molecule.

An “isolated infectious RNA molecule” refers to a composition of mattercomprising the aforementioned infectious RNA molecule purified to anydetectable degree from its naturally occurring state, if such RNAmolecule does indeed occur in nature. Likewise, an “isolatedpolynucleotide molecule” refers to a composition of matter comprising apolynucleotide molecule of the present invention purified to anydetectable degree from its naturally occurring state, if any.

For purposes of the present invention, the nucleotide sequence of asecond polynucleotide molecule (either RNA or DNA) is “homologous” tothe nucleotide sequence of a first polynucleotide molecule where thenucleotide sequence of the second polynucleotide molecule encodes thesame polyaminoacid as the nucleotide sequence of the firstpolynucleotide molecule as based on the degeneracy of the genetic code,or when it encodes a polyaminoacid that is sufficiently similar to thepolyaminoacid encoded by the nucleotide sequence of the firstpolynucleotide molecule so as to be useful in practicing the presentinvention. For purposes of the present invention, a polynucleotidemolecule is useful in practicing the present invention where it can beused as a diagnostic probe to detect the presence of the North AmericanPRRS virus in a fluid or tissue sample of an infected pig, e.g. bystandard hybridization or amplification techniques. It is to beunderstood that the polyaminoacid encoded by the nucleotide sequence ofthe polynucleotide molecule can comprise a group of two or morepolyaminoacids. Generally, the nucleotide sequence of a secondpolynucleotide molecule is homologous to the nucleotide sequence of afirst polynucleotide molecule if it has at least about 70% nucleotidesequence identity to the nucleotide sequence of the first polynucleotidemolecule as based on the BLASTN algorithm (National Center forBiotechnology Information, otherwise known as NCBI, (Bethesda, Md., USA)of the United States National Institute of Health). Preferably, ahomologous nucleotide sequence has at least about 75% nucleotidesequence identity, even more preferably at least about 85% nucleotidesequence identity. Since the genetic code is degenerate, a homologousnucleotide sequence can include any number of “silent” base changes,i.e. nucleotide substitutions that nonetheless encode the same aminoacid. A homologous nucleotide sequence can further contain non-silentmutations, i.e. base substitutions, deletions, or additions resulting inamino acid differences in the encoded polyaminoacid, so long as thesequence remains at least about 70% identical to the polyaminoacidencoded by the first nucleotide sequence or otherwise is useful forpracticing the present invention. Homologous nucleotide sequences can bedetermined by comparison of nucleotide sequences, for example by usingBLASTN, above. Alternatively, homologous nucleotide sequences can bedetermined by hybridization under selected conditions. For example, thenucleotide sequence of a second polynucleotide molecule is homologous toSEQ ID NO:1 if it hybridizes to the complement of SEQ ID NO:1 undermoderately stringent conditions, e.g., hybridization to filter-bound DNAin 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C.,and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel et al. above), orconditions which will otherwise result in hybridization of sequencesthat encode a North American PRRS virus as defined below. In anotherembodiment, a second nucleotide sequence is homologous to SEC) ID NO:1if it hybridizes to the complement of SEQ ID NO:1 under highly stringentconditions, e.g. hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7%SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.(Ausubel et al., above).

It is furthermore to be understood that the isolated polynucleotidemolecules and the isolated RNA molecules of the present inventioninclude both synthetic molecules and molecules obtained throughrecombinant techniques, such as by in vitro cloning and transcription.

As used herein, the term “PRRS” encompasses disease symptoms in swinecaused by a PRRS virus infection. Examples of such symptoms include, butare not limited to, abortion in pregnant females, and slow growth,respiratory difficulties, loss of appetite, and mortality in young pigs.As used herein, a PRRS virus that is “unable to produce PRRS” refers toa virus that can infect a pig, but which does not produce any diseasesymptoms normally associated with a PRRS infection in the pig, orproduces such symptoms, but to a lesser degree, or produces a fewernumber of such symptoms, or both.

The terms “porcine” and “swine” are used interchangeably herein andrefer to any animal that is a member of the family Suidae such as, forexample, a pig. “Mammals” include any warm-blooded vertebrates of theMammalia class, including humans.

The term “PRRS virus”, as used herein, unless otherwise indicated, meansany strain of either the North American or European PRRS viruses.

The term “field strain” or “field strains”, as used herein, unlessotherwise indicated, means any strain of either North American orEuropean PRRS virus which occurs in nature, and is infectious to a pig.

The term “North American PRRS virus” means any PRRS virus having geneticcharacteristics associated with a North American PRRS virus isolate,such as, but not limited to the PRRS virus that was first isolated inthe United States around the early 1990's (see, e.g., Collins, J. E., etal., 1992, J. Vet. Diagn. Invest. 4:117-126); North American PRRS virusisolate MN-1b (Kwang, J. et al., 1994, J. Vet. Diagn. Invest.6:293-296); the Quebec IAF-exp91 strain of PRRS (Mardassi, H. et al.,1995, Arch. Virol. 140:1405-1418); and North American PRRS virus isolateVR 2385 (Meng, X-.J et al., 1994, J. Gen. Virol. 75:1795-1801). Geneticcharacteristics refers to genomic nucleotide sequence similarity andaminoacid sequence similarity shared by North American PRRS virusstrains. For purposes of the present invention, a North American PRRSvirus is a virus that is encoded by an RNA sequence the same as orhomologous to SEQ ID NO:1, wherein the term “homologous” is as definedpreviously. Thus, strains of North American PRRS viruses have,preferably, at least about 70% genomic nucleotide sequence identity withSEQ ID NO:1, and more preferably at least about 75% genomic nucleotidesequence identity with SEQ ID NO:1, at least about 85% genomicnucleotide sequence identity with SEQ ID NO:1 being even more preferred.

The term “European PRRS virus” refers to any strain of PRRS virus havingthe genetic characteristics associated with the PRRS virus that wasfirst isolated in Europe around 1991 (see, e.g., Wensvoort, G., et al.,1991, Vet. Q. 13:121-130). “European PRRS virus” is also sometimesreferred to in the art as “Lelystad virus”.

Unless otherwise indicated, a North American PRRS virus is “useful inpracticing the present invention” if its characteristics are within thedefinition of a North American PRRS virus set forth herein. For example,a virus encoded by one of the isolated polynucleotide molecules of thepresent invention is a “North American PRRS virus useful in practicingthe present invention” if it, e.g., has genetic characteristicsassociated with a North American PRRS virus.

Other polyaminoacids are “useful in practicing the present invention”,e.g., peptides encoded by polynucleotide sequences homologous to NorthAmerican PRRS virus ORFs, if they can compensate for an RNA moleculeencoding a genetically modified PRRS virus, deficient in a geneessential for expressing functional PRRS virions, in a transfected hostcell so that functional PRRS virions can be generated by the cell.

The term “open reading frame”, or “ORF”, as used herein, means theminimal nucleotide sequence required to encode a particular PRRS virusprotein without an intervening stop codon.

Terms such as “suitable host cell” and “appropriate host cell”, unlessotherwise indicated, refer to cells into which RNA molecules (orisolated polynucleotide molecules or viral vectors comprising DNAsequences encoding such RNA molecules) of the present invention can betransformed or transfected. “Suitable host cells” for transfection withsuch RNA molecules, isolated polynucleotide molecules, or viral vectors,include mammalian, particularly porcine, and avian cells, and aredescribed in further detail below.

A “functional virion” is a virus particle that is able to enter a cellcapable of hosting a PRRS virus, and express genes of its particular RNAgenome (either an unmodified genome or a genetically modified genome asdescribed herein) within the cell. Cells capable of hosting a PRRS virusinclude porcine alveolar macrophage cells and MARC 145 monkey kidneycells. Other mammalian or avian cells, especially other porcine cells,may also serve as suitable host cells for PRRS virions.

The isolated polynucleotide molecules of the present invention encodeNorth American PRRS viruses that can be used to prepare live, killed, orattenuated vaccines using art-recognized methods for protecting swinefrom infection by a PRRS virus, as described in further detail below.These isolated polynucleotide molecules are also useful as vectors fordelivering heterologous genes into mammals, including swine, or birds,as is also described in detail below. Furthermore, these isolatedpolynucleotide molecules are useful because they can be mutated usingmolecular biology techniques to encode genetically-modified NorthAmerican PRRS viruses useful, inter alia, as vaccines for protectingswine from PRRS infection. Such genetically-modified North American PRRSviruses, as well as vaccines comprising them, are also described infurther detail below.

The term “genetically modified”, as used herein and unless otherwiseindicated, means genetically mutated, i.e. having one or morenucleotides replaced, deleted and/or added. Polynucleotide molecules canbe genetically mutated using recombinant techniques known to those ofordinary skill in the art, including by site-directed mutagenesis, or byrandom mutagenesis such as by exposure to chemical mutagens or toradiation, as known in the art. In one embodiment, genetic modificationof the North American PRRS virus of the present invention renders thevirus unable to replicate effectively, or reduces its ability toreplicate effectively, in a bird or mammal in which the wild-type virusotherwise can effectively replicate. In another embodiment, thegenetically modified North American PRRS virus of the present inventionremains able to replicate effectively in birds or mammals infectedtherewith. “Effective replication” means the ability to multiply andproduce progeny viruses (virions) in an infected animal, i.e. theability to “productively infect” an animal.

The subject invention further provides an isolated polynucleotidemolecule comprising a DNA sequence encoding an infectious RNA moleculewhich encodes a genetically modified North American PRRS virus that isunable to produce PRRS in a porcine animal, wherein the DNA sequenceencoding the infectious RNA molecule encoding said North American PRRSvirus is SEQ ID NO:1 or a sequence homologous thereto, except that itcontains one or more mutations that genetically disable the encoded PRRSvirus in its ability to produce PRRS. “Genetically disabled” means thatthe PRRS virus is unable to produce PRRS in a swine animal infectedtherewith.

The subject invention also provides an isolated polynucleotide moleculecomprising a DNA sequence encoding an infectious RNA molecule whichencodes a North American PRRS virus that is genetically modified suchthat when it infects a porcine animal it: a) is unable to produce PRRSin the animal, and b) is able to elicit an effective immunoprotectiveresponse against infection by a PRRS virus in the animal, wherein theDNA sequence encoding said North American PRRS virus is SEQ ID NO:1 or asequence homologous thereto, except that it contains one or moremutations that genetically disable the encoded PRRS virus in its abilityto produce PRRS.

The term “immune response” for purposes of this invention means theproduction of antibodies and/or cells (such as T lymphocytes) that aredirected against, or assist in the decomposition or inhibition of, aparticular antigenic epitope or particular antigenic epitopes. Thephrases “an effective immunoprotective response”, “immunoprotection”,and like terms, for purposes of the present invention, mean an immuneresponse that is directed against one or more antigenic epitopes of apathogen so as to protect against infection by the pathogen in avaccinated animal. For purposes of the present invention, protectionagainst infection by a pathogen includes not only the absoluteprevention of infection, but also any detectable reduction in the degreeor rate of infection by a pathogen, or any detectable reduction in theseverity of the disease or any symptom or condition resulting frominfection by the pathogen in the vaccinated animal as compared to anunvaccinated infected animal. An effective immunoprotective response canbe induced in animals that have not previously been infected with thepathogen and/or are not infected with the pathogen at the time ofvaccination. An effective immunoprotective response can also be inducedin an animal already infected with the pathogen at the time ofvaccination.

An “antigenic epitope” is, unless otherwise indicated, a molecule thatis able to elicit an immune response in a particular animal or species.Antigenic epitopes are proteinaceous molecules, i.e. polyaminoacidsequences, optionally comprising non-protein groups such as carbohydratemoieties and/or lipid moieties.

The term “pathogenically infecting” used herein refers to the ability ofa pathogen to infect an animal and cause a disease in the animal. As anexample, a PRRS virus is capable of pathogenically infecting a porcineanimal since it can cause PRRS in swine. However, although a PRRS virusmay be able to infect, either productively or non-productively, a birdor another mammal, such as a human, it does not pathogenically infectany animal other than a porcine animal since it does not cause anydisease in animals other than porcine animals.

The genetically modified North American PRRS viruses encoded by theabove-described isolated polynucleotide molecules are, in oneembodiment, able to elicit an effective immunoprotective responseagainst infection by a PRRS virus. Such genetically modified NorthAmerican PRRS viruses are preferably able to elicit an effectiveimmunoprotective response against any strain of PRRS viruses, includingboth European and North American strains.

The present invention provides a mutation or mutations in the isolatedpolynucleotide molecule encoding the genetically disabled North AmericanPRRS virus that are non-silent and occur in one or more open readingframes of the nucleotide sequence encoding the North American PRRSvirus; i.e., the mutation or mutations occur in one or more of thesequences within the nucleotide sequence encoding the North AmericanPRRS virus that are the same as or homologous to ORFs 1a, 1b, 2, 3, 4,5, 6, or 7 of SEQ ID NO:1. The mutation or mutations may occur in one ormore noncoding regions of the North American PRRS virus genome, such as,for example, in the leader sequence of the North American PRRS virusgenome; i.e., the mutation or mutations occur within the sequence thatis the same as or homologous to the sequence of nucleotides 1-191 of SEQID NO:1. In the same isolated polynucleotide molecule, mutations canoccur in both coding and noncoding regions.

As used herein, unless otherwise indicated, “noncoding regions” of thenucleotide sequence encoding the North American PRRS virus refer tothose sequences of RNA that are not translated into a protein and thosesequences of cDNA that encode such RNA sequences. Coding regions referto those sequences of RNA from which North American PRRS virus proteinsare expressed, and also refer to cDNA that encodes such RNA sequences.Likewise, “ORFs” refer both to RNA sequences that encode North AmericanPRRS virus proteins and to cDNA sequence encoding such RNA sequences.

Determining suitable locations for a mutation or mutations that willencode a North American PRRS virus that is genetically disabled so thatit is unable to produce PRRS yet remains able to elicit an effectiveimmunoprotective response against infection by a PRRS virus can be madebased on the SEQ ID NO:1 provided herein. One of ordinary skill canrefer to the sequence of the infectious cDNA clone of North AmericanPRRS virus provided by this invention, make sequence changes which willresult in a mutation, and test the viruses encoded thereby both fortheir ability to produce PRRS in swine, and to elicit an effectiveimmunoprotective response against infection by a PRRS virus. In sodoing, one of ordinary skill can refer to techniques known in the artand also those described and/or exemplified herein.

For example, an ORF of the sequence encoding the infectious RNA moleculeencoding the North American PRRS virus can be mutated and the resultinggenetically modified North American PRRS virus tested for its ability tocause PRRS. The ORF of a North American PRRS virus encodes proteins asfollows: ORF Ia encodes a polyprotein comprising protease function; ORF1b encodes a polyprotein comprising replicase (RNA polymerase) andhelicase functions; ORFs 2, 3, and 4 encode small membraneglycoproteins; ORF 5 encodes a major envelope glycoprotein; ORF 6encodes a nonglycosylated integral membrane protein; and ORF 7 encodes anucleocapsid protein. Genetic mutations of one or more of these ORFs canbe used in preparing the genetically modified North American PRRSviruses described infra.

The subject invention also provides an isolated polynucleotide moleculecomprising a DNA sequence encoding an infectious RNA molecule encoding aNorth American PRRS virus that is genetically modified such that itlacks at least one DNA sequence encoding a detectable antigenic epitopeof North American PRRS virus, wherein the DNA sequence encoding the RNAmolecule encoding the North American PRRS virus is SEQ ID NO:1 or asequence homologous thereto.

A “detectable antigenic epitope” is, unless otherwise indicated, amolecule that is able to elicit an immune response in a particularanimal or species, which immune response is measurable via an assay ormethod.

In a non-limiting embodiment, the detectable antigenic epitope which isdeleted is within ORF 1a or ORF 1b. In another embodiment, thedetectable antigenic epitope which is deleted is within the sequencesencoding for the nonstructural protein 2 (nsp2), which is part of ORF1a. In a separate embodiment, the detectable antigenic epitope which isdeleted is within the hyperviariable C-terminal portion of nsp2. In afurther embodiment, the detectable antigenic epitope which is deleted,or lacking, is the DNA sequence encoding any 10 aa polypeptides orlonger, up to 400 aa length polypeptides, where the DNA sequence thatmay be deleted is the DNA that encodes any amino acids from 616 to 752of the hypervariable region of nsp2. This region, from 616 to 752 ofnsp2, is the conserved sub-region within the hypervariable region ofnsp2.

One example of such a deletion is Example XI. In Example XI a deletionis described which results in the lack of a DNA sequence encoding aminoacids 628 through 759 of nsp2. This deletion is within the regionencoding the hypervariable C-terminal portion of nsp2. This Example XIdeletion, which lacks the DNA sequence encoding amino acids from 628 to759 in the hypervariable region is considered as being taken from theregion of the DNA sequence encoding amino acids 616 to 752 in thehypervariable region because the region begins in the region of the DNAsequence encoding amino acids 616 to 752 in the hypervariable region.The deleted region can extend beyond the conserved sub-region in eitherdirection, but the entire deleted region should be no greater than theamount of DNA that encodes for a polypeptide of about 400 aa. Table 1shows the relative positions of some of the nucleotides and amino acidsin relation to certain regions of the genome for SEQ. ID. NO. 1.

TABLE 1 Genome coordinates of nsp2 regions based on SEQ ID NO. 1.Amino acid Genome position Amino acid range range Landmark (nucleotides)(w/in ORF1a) (w/in nsp2) Entire 1-15,450 NA NA genome(1-15,395 w/o polyA tail) ORF1a 192-7685 1-2497 NA (7491 nt) (2497 aa)ATGTCT . . . TGCTAG MSGIL . . . QCLNC Nsp2 1341-4262 384-1357 1-974(2922 nt) (974 aa) (974 aa) GCTGGA . . . CTGGGC AGKRA . . . GRLLGAGKRA . . . GRLLG Hypervariable 2721-3980 844-1263 461-880region of nsp2 (1260 nt) (420 aa) (420 aa) AGATCT . . . CTCTTTRSDYG . . . LFCLF RSDYG . . . LFCLF Conserved 3186-3596 999-1135 616-752subregion w/in (411 nt) (137 aa) (137 aa) hypervariable regionTCATCA . . . CGCATC SSSSS . . . DIPRI SSSSS . . . DIPRI Deletion in3222-3617 1011-1142 628-759 Example XI (396 nt) (132 aa) (132 aa)CGCCCA . . . AATACC RPKYS . . . KIENT RPKYS . . . KIENT

Such deletions may be anywhere within the hypervariable region,including the region of the DNA encoding the conserved sub-region ofamino acids from 616 to 752 in nsp2, and such deletions may extend intothe flanking regions as well, provided the DNA encodes at least 10 aminoacids from the conserved sub-region or from other locations in thehypervariable region.

In a further embodiment, the detectable antigenic epitope which isremoved is taken from the DNA sequence encoding amino acids 628 through759 of nsp2, which is both within the conserved sub-region and withinthe region encoding the hypervariable C-terminal portion of nsp2.

In a further embodiment, the detectable antigenic epitope which isdeleted is taken from the DNA sequence encoding polypeptides as short asten amino acids, or as long as the entire hypervariable region of nsp2which can extend up to 400 amino acids. Specifically the region may befrom 10 to 100 aa in length, from 100 to 150, from 150 to 200, from 200to 300 or from 300 to 400 amino acids in length. For example, thedeleted DNA can encode the peptide that comprises amino acids 1-10 ofthe hypervariable region of the nsp2 peptide. The peptide can be aminoacids 2-11 of the hypervariable region. The peptide can be amino acids3-12 of the hypervariable region. Peptides are selected in this fashionby progressively “walking” down the amino acid sequence of thehypervariable region of nsp2, all of the way through the hypervariableregion, more particularly through the region of the DNA encoding theconserved subregion of amino acids from 616 to 752 of nsp2 within thehypervariable region, and it may extend into the flanking regions. Aspecific example of this is Example XI where the deleted region is theDNA sequence encoding amino acids 628 through 759 in the hypervariableregion.

In addition to 10-mer peptides, the deleted fragment can be a DNAencoding 11 amino acid residues in length, beginning with a fragmentcomprising amino acid 1-11 of the hypervariable region, and progressingto the C-terminal end of the hypervariable region. Similarly, thefragment can be DNA encoding 12, 13, 14, 15, 16, 17, 18, 19, 20 or moreresidues in length, up to 400 residues in length, and such peptides arereadily identifiable by beginning at residue 1 and “walking” down theamino acid sequence of the corresponding hypervariable region of nsp2.Such a strategy can be applied to the corresponding hypervariable regionof the nsp2 sequence from any strain of PRRS virus.

The subject invention also provides an isolated polynucleotide moleculecomprising a DNA sequence encoding an infectious RNA molecule encoding aNorth American PRRS virus that is genetically modified such that itlacks at least one DNA sequence encoding a detectable antigenic epitopeof North American PRRS virus, wherein it further comprises one or moreheterologous antigenic epitopes, wherein the DNA sequence encoding theRNA molecule encoding the North American PRRS virus is SEC) ID NO:1 or asequence homologous thereto, and further comprising one or moreadditional nucleotide sequences that each encode a heterologousantigenic epitope, and wherein each heterologous antigenic epitope iscapable of inducing an effective immunoprotective response against aparticular pathogen in a mammal or a bird.

A pathogen against which an effective immunoprotective response can beinduced by means of the above recited aspect of the present invention isany pathogen, such as a virus, bacteria, fungus, or protozoan, capableof causing a disease in a mammal or bird, which pathogen comprises orhas associated therewith one or more antigenic epitopes which can beused to induce an effective immunoprotective response against thepathogen in the mammal or bird.

The term “heterologous antigenic epitope” for purposes of the presentinvention means an antigenic epitope, as defined above, not normallyfound in a wild-type North American PRRS virus. A nucleotide sequenceencoding a heterologous antigenic epitope can be inserted into a NorthAmerican PRRS viral genome using known recombinant techniques. Antigenicepitopes useful as heterologous antigenic epitopes for the presentinvention include additional North American PRRS virus antigenicepitopes, antigenic epitopes from European PRRS viruses, antigenicepitopes from swine pathogens other than PRRS viruses, or antigenicepitopes from pathogens that pathogenically infect birds or mammalsother than swine, including humans. Sequences encoding such antigenicepitopes are known in the art or are provided herein. For example, asecond North American PRRS virus envelope protein, encoded by NorthAmerican PRRS ORF 5 described herein, can be inserted into a DNAsequence encoding an RNA molecule encoding a North American PRRS virusof the present invention to generate a genetically modified NorthAmerican PRRS virus comprising an additional envelope protein as aheterologous antigenic epitope. Such a genetically modified NorthAmerican PRRS virus can be used to induce a more effectiveimmunoprotective response against PRRS viruses in a porcine animalvaccinated therewith.

Examples of an antigenic epitope from a swine pathogen other than aNorth American PRRS virus include, but are not limited to, an antigenicepitope from a swine pathogen selected from the group consisting ofEuropean PRRS, porcine parvovirus, porcine circovirus, a porcinerotavirus, swine influenza, pseudorabies virus, transmissiblegastroenteritis virus, porcine respiratory coronavirus, classical swinefever virus, African swine fever virus, encephalomyocarditis virus,porcine paramyxovirus, Actinobacillus pleuropneumoni, Bacillus anthraci,Bordetella bronchiseptica, Clostridium haemolyticum, Clostridiumperfringens, Clostridium tetani, Escherichia coli, Erysipelothrixrhusiopathiae, Haemophilus parasuis, Leptospira spp., Mycoplasmahyopneumoniae, Mycoplasma hyorhinis, Pasteurella haemolytica,Pasteurella multocida, Salmonella choleraesuis, Salmonella typhimurium,Streptococcus equismilis, and Streptococcus suis. Nucleotide sequencesencoding antigenic epitopes from the aforementioned swine pathogens areknown in the art and can be obtained from public gene databases such asGenBank (http://www.ncbi.nlm.nih.gov/Web/Genbank/index.html) provided byNCBI.

If the heterologous antigenic epitopes are antigenic epitopes from oneor more other swine pathogens, then the isolated polynucleotide moleculecan further contain one or more mutations that genetically disable theencoded PRRS virus in its ability to produce PRRS. Such isolatedpolynucleotide molecules and the viruses they encode are useful forpreparing vaccines for protecting swine against the swine pathogen orpathogens from which the heterologous antigenic epitopes are derived.

The present invention provides a genetically modified North AmericanPRRS virus able to elicit an effective immunoprotective response againstinfection by a PRRS virus in a porcine animal. Such isolatedpolynucleotide molecules and the viruses they encode are useful forpreparing dual-function vaccines for protecting swine against infectionby both a North American PRRS virus and the swine pathogen or pathogensfrom which the heterologous antigenic epitopes are derived.

The isolated polynucleotide molecules of the present inventioncomprising nucleotide sequences encoding heterologous antigenic epitopescan be prepared as described above based on the sequence encoding aNorth American PRRS virus described herein using known techniques inmolecular biology.

In one embodiment, the additional nucleotide sequences that each encodea heterologous antigenic epitope are inserted into the DNA sequenceencoding an infectious RNA molecule encoding a North American PRRS viruswithin an open reading frame encoding for a PRRS virus protein. In anon-limiting embodiment, the additional nucleotide sequences areinserted in ORF 1a or ORF 1b. In another embodiment, the additionalnucleotide sequences are inserted within sequences encoding for thenonstructural protein 2 (nsp2), which is part of ORF 1a. In a separateembodiment, the additional nucleotide sequences are inserted within theregion encoding the hyperviariable C-terminal portion of nsp2. In afurther embodiment, the additional nucleotide sequences are insertedbetween the DNA sequence encoding amino acids 628 and 759 in thehypervariable region in the nonstructural protein 2 coding region ofORF1a within said DNA sequence, which is within the region encoding thehypervariable C-terminal portion of nsp2. See FIG. 4.

The hypervariable region of nsp2 corresponds approximately to genomecoordinates 2720-3980 in P129. See FIG. 4.

In a separate embodiment, the additional nucleotide sequences that eachencode a heterologous antigenic epitope are inserted into the DNAsequence encoding an infectious RNA molecule encoding a North AmericanPRRS virus within the sequences encoding any of ORFs 1a, 1b, 2, 3, 4, 5,6, or 7. See FIG. 4.

In another embodiment, a heterologous antigenic epitope of thegenetically modified North American PRRS virus of the present inventionis a detectable antigenic epitope. Such isolated polynucleotidemolecules and the North American PRRS viruses they encode are useful,inter alia, for studying PRRS infections in swine, determiningsuccessfully vaccinated swine, and/or for distinguishing vaccinatedswine from swine infected by a wild-type PRRS virus. Preferably, suchisolated polynucleotide molecules further contain one or more mutationsthat genetically disable the encoded PRRS virus in its ability toproduce PRRS, and more preferably are able to elicit an effectiveimmunoprotective response in a porcine animal against infection by aPRRS virus.

Heterologous antigenic epitopes that are detectable, and the sequencesthat encode them, are known in the art. Techniques for detecting suchantigenic epitopes are also known in the art and include serologicaldetection of antibody specific to the heterologous antigenic epitope bymeans of, for example, Western blot, ELISA, or fluorescently labeledantibodies capable of binding to the antibodies specific to theheterologous antigenic epitope. Techniques for serological detectionuseful in practicing the present invention can be found in textsrecognized in the art, such as Coligan, J. E., et al. (eds), 1998,Current Protocols in Immunology, John Willey & Sons, Inc., which ishereby incorporated by reference in its entirety. Alternatively, theheterologous antigenic epitope itself can be detected by, for example,contacting samples that potentially comprise the antigenic epitope withfluorescently-labeled antibodies or radioactively-labeled antibodiesthat specifically bind to the antigenic epitopes.

The present invention further provides an isolated polynucleotidemolecule comprising a DNA sequence encoding an infectious RNA moleculewhich encodes a genetically modified North American PRRS virus thatdetectably lacks a North American PRRS virus antigenic epitope, whereinthe DNA sequence encoding the RNA molecule encoding the North AmericanPRRS virus is SEQ ID NO:1 or a sequence homologous thereto, except thatit lacks one or more nucleotide sequences encoding a detectable NorthAmerican PRRS virus antigenic epitope. Such isolated polynucleotidemolecules are useful for distinguishing between swine infected with arecombinant North American PRRS virus of the present invention and swineinfected with a wild-type PRRS virus. For example, animals vaccinatedwith killed, live or attenuated North American PRRS virus encoded bysuch an isolated polynucleotide molecule can be distinguished fromanimals infected with wild-type PRRS based on the absence of antibodiesspecific to the missing antigenic epitope, or based on the absence ofthe antigenic epitope itself: If antibodies specific to the missingantigenic epitope, or if the antigenic epitope itself, are detected inthe animal, then the animal was exposed to and infected by a wild-typePRRS virus. Means for detecting antigenic epitopes and antibodiesspecific thereto are known in the art, as discussed above. Preferably,such an isolated polynucleotide molecule further contains one or moremutations that genetically disable the encoded PRRS virus in its abilityto produce PRRS. More preferably, the encoded virus remains able toelicit an effective immunoprotective response against infection by aPRRS virus.

B. Vaccines and Uses Thereof.

The present invention also provides vaccines comprising North AmericanPRRS viruses, including genetically modified North American PRRS virusesdisabled in their ability to produce PRRS in a swine animal as describedherein; infectious RNA molecules and plasmids encoding such NorthAmerican PRRS viruses as described herein; and viral vectors encodingsuch North American PRRS viruses and isolated RNA molecules as describedherein.

In one embodiment, the subject invention provides a vaccine comprising agenetically modified North American PRRS virus which lacks at least onedetectable antigenic epitope as described herein, an infectious RNAmolecule encoding such a genetically modified North American PRRS virus,a plasmid as described herein encoding such a genetically modified NorthAmerican PRRS virus, or a viral vector encoding such a geneticallymodified North American PRRS virus, and a carrier acceptable forpharmaceutical or veterinary use, in an amount effective to elicit animmunoprotective response against PRRS virus infection in the porcineanimal.

In another embodiment, the subject invention provides a vaccinecomprising a genetically modified North American PRRS virus comprisingone or more detectable heterologous antigenic epitopes as describedherein, an infectious RNA molecule encoding such a genetically modifiedNorth American PRRS virus, a plasmid as described herein encoding such agenetically modified North American PRRS virus, or a viral vectorencoding such a genetically modified North American PRRS virus, and acarrier acceptable for pharmaceutical or veterinary use.

Such vaccines can be used to protect from infection a mammal or a birdcapable of being pathogenically infected by the pathogen or pathogensfrom which the detectable heterologous antigenic epitope(s) are derived.If such a vaccine comprises the genetically modified North American PRRSvirus, the genetic modification of the North American PRRS viruspreferably renders the virus unable to cause PRRS in swine. Thus, suchwould provide a dual-vaccine for swine, protecting swine from infectionby the swine pathogen or pathogens from which the heterologous antigenicepitope(s) are derived as well as from infection by a PRRS virus. If thevaccine comprises an infectious RNA molecule or a plasmid encoding agenetically-modified North American PRRS virus comprising one or moreheterologous antigenic epitopes from another swine pathogen, then thesequence encoding the infectious RNA molecule encoding the geneticallymodified PRRS virus preferably comprises one or more further mutationsthat genetically disable the encoded North American PRRS virus so thatit is unable to cause PRRS. In another preferred embodiment, the encodedgenetically modified, disabled North American PRRS virus is able toelicit an immunoprotective response against a PRRS infection in a swineanimal, thus providing a dual-vaccine for swine, able to protect swinefrom infection by the swine pathogen or pathogens from which theheterologous antigenic epitope(s) are derived as well as from infectionby a PRRS virus. All of these vaccines also further comprise a carrieracceptable for veterinary use.

Vaccines of the present invention can be formulated following acceptedconvention to include acceptable carriers for animals, including humans(if applicable), such as standard buffers, stabilizers, diluents,preservatives, and/or solubilizers, and can also be formulated tofacilitate sustained release. Diluents include water, saline, dextrose,ethanol, glycerol, and the like. Additives for isotonicity includesodium chloride, dextrose, mannitol, sorbitol, and lactose, amongothers. Stabilizers include albumin, among others. Other suitablevaccine vehicles and additives, including those that are particularlyuseful in formulating modified live vaccines, are known or will beapparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Science, 18th ed., 1990, Mack Publishing, which isincorporated herein by reference.

Vaccines of the present invention may further comprise one or moreadditional immunomodulatory components such as, e.g., an adjuvant orcytokine, among others. Non-limiting examples of adjuvants that can beused in the vaccine of the present invention include the RIBI adjuvantsystem (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminumhydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as,e.g., Freund's complete and incomplete adjuvants, Block copolymer(CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.),SAF-M (Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil Aor other saponin fraction, monophosphoryl lipid A, and Avridinelipid-amine adjuvant. Non-limiting examples of oil-in-water emulsionsuseful in the vaccine of the invention include modified SEAM62 and SEAM1/2 formulations. Modified SEAM62 is an oil-in-water emulsion containing5% (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICISurfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5%(v/v) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol, and 0.5% (v/v)lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising 5%(v/v) squalene, 1% (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween 80detergent, 2.5% (v/v) ethanol, 100 μg/ml Quil A, and 50 μg/mlcholesterol. Other immunomodulatory agents that can be included in thevaccine include, e.g., one or more interleukins, interferons, or otherknown cytokines.

Vaccines of the present invention can optionally be formulated forsustained release of the virus, infectious RNA molecule, plasmid, orviral vector of the present invention. Examples of such sustainedrelease formulations include virus, infectious RNA molecule, plasmid, orviral vector in combination with composites of biocompatible polymers,such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid),methylcellulose, hyaluronic acid, collagen and the like. The structure,selection and use of degradable polymers in drug delivery vehicles havebeen reviewed in several publications, including A. Domb et al., 1992,Polymers for Advanced Technologies 3: 279-292, which is incorporatedherein by reference. Additional guidance in selecting and using polymersin pharmaceutical formulations can be found in texts known in the art,for example M. Chasin and R. Langer (eds), 1990, “Biodegradable Polymersas Drug Delivery Systems” in: Drugs and the Pharmaceutical Sciences,Vol. 45, M. Dekker, NY, which is also incorporated herein by reference.Alternatively, or additionally, the virus, plasmid, or viral vector canbe microencapsulated to improve administration and efficacy. Methods formicroencapsulating antigens are well-known in the art, and includetechniques described, e.g., in U.S. Pat. No. 3,137,631; U.S. Pat. No.3,959,457; U.S. Pat. No. 4,205,060; U.S. Pat. No. 4,606,940; U.S. Pat.No. 4,744,933; U.S. Pat. No. 5,132,117; and International PatentPublication WO 95/28227, all of which are incorporated herein byreference.

Liposomes can also be used to provide for the sustained release ofvirus, plasmid, or viral vector. Details concerning how to make and useliposomal formulations can be found in, among other places, U.S. Pat.No. 4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,921,706; U.S.Pat. No. 4,927,637; U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050;and U.S. Pat. No. 5,009,956, all of which are incorporated herein byreference.

An effective amount of any of the above-described vaccines can bedetermined by conventional means, starting with a low dose of virus,plasmid or viral vector, and then increasing the dosage while monitoringthe effects. An effective amount may be obtained after a singleadministration of a vaccine or after multiple administrations of avaccine. Known factors can be taken into consideration when determiningan optimal dose per animal. These include the species, size, age andgeneral condition of the animal, the presence of other drugs in theanimal, and the like. The actual dosage is preferably chosen afterconsideration of the results from other animal studies.

One method of detecting whether an adequate immune response has beenachieved is to determine seroconversion and antibody titer in the animalafter vaccination. The timing of vaccination and the number of boosters,if any, will preferably be determined by a doctor or veterinarian basedon analysis of all relevant factors, some of which are described above.

The effective dose amount of virus, infectious RNA molecule, plasmid, orviral vector, of the present invention can be determined using knowntechniques, taking into account factors that can be determined by one ofordinary skill in the art such as the weight of the animal to bevaccinated. The dose amount of virus of the present invention in avaccine of the present invention preferably ranges from about 10¹ toabout 10⁹ pfu (plaque forming units), more preferably from about 10² toabout 10⁸ pfu, and most preferably from about 10³ to about 10⁷ pfu. Thedose amount of a plasmid of the present invention in a vaccine of thepresent invention preferably ranges from about 0.1 μg to about 100 mg,more preferably from about 1 μg to about 10 mg, even more preferablyfrom about 10 μg to about 1 mg. The dose amount of an infectious RNAmolecule of the present invention in a vaccine of the present inventionpreferably ranges from about 0.1 μg to about 100 mg, more preferablyfrom about 1 μg to about 10 mg, even more preferably from about 10 μg toabout 1 mg. The dose amount of a viral vector of the present inventionin a vaccine of the present invention preferably ranges from about 10¹pfu to about 10⁹ pfu, more preferably from about 10² pfu to about 10⁸pfu, and even more preferably from about 10³ to about 10⁷ pfu. Asuitable dosage size ranges from about 0.5 ml to about 10 ml, and morepreferably from about 1 ml to about 5 ml.

It is to be understood that the term “North American PRRS viruses of thepresent invention” and like terms, unless otherwise indicated, includeany of the genetically modified North American PRRS viruses describedherein as well as the unmodified North American PRRS virus describedherein encoded by SEC) ID NO:1 or a sequence homologous thereto.

C. Diagnostic Kits

The present invention also provides diagnostic kits. The kit can bevaluable for differentiating between porcine animals naturally infectedwith a field strain of a PRRS virus and porcine animals vaccinated withany of the vaccines described herein. The kits can also be of valuebecause animals potentially infected with field strains of PRRS viruscan be detected prior to the existencein the absence of clinicalsymptoms and removed from the herd, or kept in isolation away from naïveor vaccinated animals. The kits include reagents for analyzing a samplefrom a porcine animal for the presence of antibodies to a PRRS virus.Diagnostic kits of the present invention can include as a component apeptide or peptides which comprise all or a portion of a proteinsequence which is no longer present in a genetically modified NorthAmerican PRRS virus which may optionally comprise one or more detectableheterologous antigenic epitopes. Kits of the present invention canalternatively include as a component a peptide which is a fusionprotein. The term “fusion peptide” or “fusion protein” for purposes ofthe present invention means a single polypeptide chain consisting of atleast a portion of a PRRS virus protein and a heterologous peptide orprotein.

D. Plasmids Encoding a North American PRRS Virus or a GeneticallyModified North American PRRS Virus.

The present invention also provides any of the above-described isolatedpolynucleotide molecules in the form of a plasmid capable of expressingthe North American PRRS virus encoded thereby.

Plasmids of the present invention can express the encoded North AmericanPRRS virus outside of a living organism, to produce North American PRRSviruses of the invention useful, inter alia, for preparing vaccines. Inone embodiment, a plasmid of the present invention capable of expressinga North American PRRS virus outside of a living organism is a plasmidwherein transcription of viral RNA therefrom occurs in vitro (i.e.extracellularly); the resulting viral RNA molecule is transfected into asuitable host cell using known mechanisms of transfection, such aselectroporation, lipofection (in some cases using a commerciallyavailable reagent, such as Lipofectin™ (Life Technologies Inc.,Rockville, Md., USA)), or DEAE dextran mediated transfection. Othermethods of transfection are known in the art and can be employed in thepresent invention. An example of such a plasmid for in vitrotranscription of North American PRRS viral RNA is the plasmid pT7P129A(ATCC Accession No. 203488). Any promoter useful for in vitrotranscription can be used in such plasmids of this invention. T7 is onesuch promoter, but other promoters can be used, such as an SP6 promoteror a T3 promoter. The sequences of such promoters can be artificiallysynthesized or cloned from commercially available plasmids. Suitableplasmids for preparing such plasmids capable of expressing NorthAmerican PRRS virus include, but are not limited to, general purposecloning vector plasmids such as pCR2.1 (Invitrogen, Carlsbad, Calif.,USA), pBR322, and pUC18. A nucleotide sequence of the present inventionencoding the North American PRRS virus can be inserted into any of theseplasmids using known recombinant techniques. Other plasmids into whichthe polynucleotide molecules of the present invention can be insertedwill be recognized by those of ordinary skill in the art.

Suitable conditions for in vitro transcription of viral RNA from any ofthe above-described recombinant plasmids comprising a DNA sequenceencoding an infectious RNA molecule encoding a North American PRRS virusdepends on the type of plasmid, for example, its particular promoter,and can be ascertained by one of ordinary skill in the art. For example,if a plasmid of the present invention is based on a pCR2.1 plasmidcomprising a T7 promoter, then an example of suitable conditions for invitro transcription includes reacting the plasmid with T7 RNA polymeraseand ribonucleotides in a standard buffer and incubating the reaction at37° C. for about 30 minutes. In some cases, commercial kits areavailable for transcribing RNA from a particular plasmid, and such kitscan be used in the present invention. The reaction mixture followingtranscription can be directly transfected into a suitable host cellwithout purification, or the transcribed North American PRRS virus RNAcan be purified by known RNA purification techniques, for example byorganic (e.g. phenol) extraction and alcohol (e.g. ethanol orisopropanol) precipitation, prior to transfection.

Transcription of the DNA sequence encoding an infectious RNA moleculeencoding a North American PRRS virus, and further comprising one or moreadditional nucleotide sequences that each encode a heterologousantigenic epitope, results in the formation of a genomic and subgenomicRNA molecules. In one embodiment, at least one heterologous antigenicepitope is translated directly from the genomic RNA molecule. In anotherembodiment, at least one heterologous antigenic epitope is translatedfrom a subgenomic RNA molecule that is synthesized from the genomic RNA.

The term “translation” for purposes of the present invention means thesynthesis of protein from nucleotides such as an RNA molecule. In oneembodiment, translation of the genomic or a subgenomic RNA moleculeencoding a heterologous antigenic epitope results in synthesis of apeptide or protein consisting of the heterologous antigenic epitopeitself. In another embodiment, translation of the genomic or asubgenomic RNA molecule results in synthesis of a fusion peptide orprotein. The term “fusion peptide or protein” for purposes of thepresent invention means a single polypeptide chain consisting of atleast a portion of a PRRS virus protein and at least one heterologousantigenic epitope.

Practically any mammalian or avian cell culture can be transfected withthe North American PRRS virus RNA obtained as described above in orderto generate a first round of North American PRRS virions. An example ofcells which one might find particularly useful because of their readyavailability and ease of use are BHK (baby hamster kidney) cells.However, if one wishes to generate a cell culture capable of sustainedproduction of North American PRRS virions, then porcine alveolarmacrophage cells or MARC-145 cells (Kim, H. S., et al., supra) arepreferred since these cells excrete high levels of new generation PRRSvirions subsequent to PRRS virus infection. Other cell lines derivedfrom the MA-104 cell line may also be used for sustained generation ofNorth American PRRS virions of the present invention. Primary porcinealveolar macrophage cells can be obtained by lung lavages from pigs, andthe MARC-145 monkey kidney cell line can be obtained from the NationalVeterinary Services Laboratories otherwise known as NVSL (Ames, Iowa,USA).

In another embodiment, a plasmid capable of expressing a North AmericanPRRS virus of the present invention outside of a living organism is aplasmid which is transfected into a suitable host cell, for example byelectroporation or lipofection, transcription of the infectious RNAmolecule and expression of the North American PRRS virus therefromoccurring within the transfected host cell. The transfected host celltherefore generates North American PRRS virions. Such a completelycellular method has heretofore never been disclosed or suggested for anyvirus within the order of Nidovirales. Because of possible crypticsplicing and termination sequences present in the RNA genome of virusesof the Nidovirales order, a completely cellular method of expressing aNidovirales virus was believed unlikely. Cryptic sequences include RNAsplice donor and splice acceptor sequences, which could causeinappropriate splicing of the RNA transcript, as well as polyadenylationsequences, which could cause premature termination by the cellular RNApolymerase II. The present invention demonstrates, however, that thepresence of such sequences in a plasmid comprising a cDNA clone of aNidovirus does not prevent the plasmid's ability to express theNidovirus when the plasmid is directly transfected into a suitable hostcell.

Accordingly, the subject invention also provides plasmids and acompletely cellular method for expressing a Nidovirales virus, whereinthe plasmid comprises: a) a DNA sequence encoding an infectious RNAmolecule encoding the Nidovirales virus; and b) a promoter capable oftranscribing said encoding sequence in a cell, wherein said promoter isin operative association with the DNA sequence encoding the infectiousRNA molecule. The method comprises transfecting a suitable host cellwith such a plasmid, subjecting the transfected host cell to conditionssuitable for expression of gene sequences transfected therein, andcollecting the expressed Nidovirales virus therefrom. An example of aplasmid suitable for completely cellular expression of North AmericanPRRS virus outside of a living organism is the plasmid pCMV-S-P129 (ATCCAccession No. 203489). In a preferred embodiment, the promoter of such aplasmid is a CMV promoter. In a preferred embodiment, a plasmid of theinvention suitable for a completely cellular method of expressing aNidovirales virus comprises a eukaryotic promoter, such as a CMVpromoter, immediately upstream and adjacent to the nucleotide sequenceencoding the Nidovirales virus. In a preferred embodiment, thenucleotide sequence encoding the Nidovirales virus encodes a PRRS virus,either European or North American. Other examples of Nidovirales virusesthat can be expressed by means of the above-described completelycellular method include other Arteriviruses such as, equine arteritisvirus, lactate dehydrogenase-elevating virus, and simian haemorrhagicfever virus; viruses that are members of the genus Coronaviridae, suchas, but not limited to, feline infectious peritinitis virus, felineenteric coronavirus, canine coronavirus, bovine coronavirus, porcinerespiratory coronavirus, turkey coronavirus, porcine transmissiblegastroenteritis virus, human coronavirus, murine hepatitis virus, andavian infectious bronchitis virus; and members of the genus Toroviridae,such as, but not limited to, Berne virus, Breda virus, and humantorovirus. Thus, plasmids suitable for completely cellular expressioncomprising a nucleotide sequence encoding one of these viruses are alsoencompassed by the present invention.

Suitable plasmids that can be used to prepare recombinant plasmids ofthe present invention for completely cellular expression outside of aliving organism of a Nidovirales virus, such as a PRRS virus, includevirtually any plasmid useful for transfection and expression ineukaryotic cells. An examples of a plasmid suitable for preparingrecombinant plasmids of the present invention for completely cellularexpression of a Nidovirales virus is the plasmid pCMVbeta (Clontech,Palo Alto, Calif., USA). Other plasmids which are able to transfect andexpress genes in eukaryotic cells which can be used to prepare plasmidsof the present invention include, but are not limited to, pcDNA3.1,pRc/RSV, and pZeoSV2 (all from Invitrogen); and pCMV-Sport3 andpSV-Sport1 (both from Life Technologies Inc.). However, almost anyeukaryotic expression vector will work for the present invention.Constructs based on cosmids can also be used for completely cellular exvivo expression of a Nidovirales virus.

Suitable host cells for the completely cellular method of the presentinvention for expressing PRRS virus include porcine alveolar macrophagecells and the MARC-145 cells, described above. Methods of transfectingthese cells with a plasmid are basically the same as those methods fortransfecting cells with viral RNA described above. Such methods include,but are not limited to, electroporation, lipofection, DEAE dextranmediated transfection, and calcium phosphate coprecipitation.

Once host cells, such as porcine alveolar macrophage cells or a MARC-145cells, have been transfected according to the subject invention, eitherwith viral RNA or with a plasmid comprising a nucleotide sequenceencoding a virus, then the cells can be frozen at about −80° C. or belowfor storage for up to several years. For longer periods of time, i.e.decades, storage in liquid nitrogen is preferred. If relatively frequentuse of the encoded virus is envisioned, then cells hosting the virus canalso be maintained (unfrozen) in culture using known techniques, forshorter periods of time. Moreover, viral particles excreted by suchcells can be stored frozen at about −80° C. or below as a source ofvirus. Transfection of such cell lines with the polynucleotide moleculeencoding the virus can be confirmed if desired, for example, by testingexhausted medium excreted by the cell line for a PRRS virus antigenusing an immunofluorescent antibody test. Antibodies which are specificfor PRRS virus antigens are known in the art (see, e.g., Collins, E. J.,et al., WO 93/03760 Mar. 4, 1993).

In another embodiment, a plasmid of the present invention comprising anucleotide sequence encoding a North American PRRS virus is suitable forin vivo expression of the North American PRRS virus, i.e. expression ina living organism. Plasmids which can be used for preparing recombinantplasmids for in vivo expression of a North American PRRS virus include,but are not limited to the plasmids capable of transfecting eukaryoticcells described above, such as pCMVbeta.

Animals that can be transfected with plasmids of the present inventioninclude mammals and birds. If the animal is other than a porcine animal,for example, a mallard duck, then the plasmid can comprise a nucleotidesequence encoding a North American PRRS virus comprising furtherantigenic epitopes from pathogens which are capable of pathogenicallyinfecting the animal; in such a case, the plasmid will encode a NorthAmerican PRRS virus serving as a vector for transporting epitopes intothe animal. If the animal is a porcine animal, then the plasmid canusefully encode any of the North American PRRS viruses described herein,including the genetically-modified North American PRRS viruses describedherein.

E. Viral Vectors Encoding a North American PRRS Virus, Including ViralVectors Encoding Genetically Modified North American PRRS Viruses:

The present invention also provides viral vectors comprising a DNAsequence encoding an infectious RNA molecule encoding any of the NorthAmerican PRRS viruses described herein, including thegenetically-modified North American PRRS viruses described herein. Suchviral vectors are useful for transfecting eukaryotic cells forproduction of PRRS viruses of the present invention outside of a livingorganism, or for transfecting swine, or other mammals, or avians, withthe sequence encoding the North American PRRS virus, for in vivoexpression of the North American PRRS virus therein.

Some examples of viruses that can be used as vectors for preparing theviral vectors of the present invention include, but are not limited to,swine viruses such as, but not limited to, swine pox virus, pseudorabiesvirus, or African swine fever virus. Such swine viruses can be obtainedfrom The National Veterinary Services Laboratories (Ames, Iowa, USA) ofthe United States Department of Agriculture; the American Type CultureCollection, otherwise known as the ATCC (Manassas, Va., USA); and otherknown sources. Recombinant viral vectors based on suitable swine virusessuch as the aforementioned swine viruses are useful for transfectingswine animals with a nucleotide sequence encoding a North American PRRSvirus of the present invention.

Viral vectors comprising a DNA sequence encoding an infectious RNAmolecule encoding a North American PRRS virus of the present inventionbased on these and other viruses can be prepared using known recombinanttechniques described in texts such as those cited previously in thisapplication.

F. Transfected Host cells Encoding or a Genetically Modified NorthAmerican PRRS Viruses.

The present invention also provides transfected host cells that comprisea DNA sequence encoding an infectious RNA molecule encoding any of theNorth American PRRS viruses described herein, including thegenetically-modified North American PRRS viruses described herein, whichtransfected host cells are capable of expressing the North American PRRSvirus. Such transfected host cells are useful for producing NorthAmerican PRRS viruses of the present invention. Examples of transfectedhost cells of the present invention include the transfected porcinealveolar macrophage cells and the transfected MARC-145 cells describedabove.

Other transfected host cells of the invention include, but are notlimited to, transfected MA-104 cells and other derivatives of MA-104cells that are transfected; transfected Baby Hamster Kidney (BHK) cells;transfected Chinese Hamster Ovary (CHO) cells; and African Green Monkeykidney cells other than MA-104 cells or MARC-145 cells, such as VEROcells; that are transfected.

G. North American PRRS Viruses, Including Genetically Modified NorthAmerican PRRS Viruses:

The present invention also provides North American PRRS viruses asdescribed herein, including genetically-modified North American PRRSviruses as described herein, expressed and/or encoded by any of theabove-described isolated polynucleotide molecules, RNA molecules,plasmids, viral vectors, or transfected host cells.

In certain situations, for example where the North American PRRS virusis to be used in a vaccine for swine and the North American PRRS virushas not been genetically modified as described above so as to be unableto cause PRRS, it is desirable to treat the North American PRRS virus,for example by inactivating or attenuating it, so that it is unable tocause PRRS in swine to which it is administered. Known methods can beused to inactivate a North American PRRS virus of the present inventionso that it is unable to cause PRRS in an animal. Examples of suchmethods include, but are not limited to, treatment with formaldehyde,BEI (binary ethyleneimine), or BPL (beta-propiolactone). Methods ofattenuation are also known in the art, and such methods can be used toattenuate a North American PRRS virus of the present invention. A NorthAmerican PRRS virus of the present invention can, for example, beattenuated by serial passage in cell culture.

If a North American PRRS virus of the present invention is for use in ananimal other than a porcine animal, or if it has been geneticallymodified as described herein so that it is unable to produce PRRS in aporcine animal, then it is not necessary to treat the virus as describedin the preceding paragraph prior to using it in a vaccine.

The following examples are provided to merely illustrate aspects of thesubject invention. They are not intended, and should not be construed,to limit the invention set forth in the claims and more fully describedherein.

EXAMPLES Example I Preparation of an Infectious cDNA Clone of a NorthAmerican PRRS Virus Isolate

Source of PRRS virus and MARC-145 cells: A North American PRRS virusisolate designated P129 was obtained from Drs. Gregory W. Stevenson,William G. Van Alstine, and Charles L. Kanitz of Purdue University'sAnimal Disease Diagnostic Laboratory in West Lafayette, Ind. The P129virus was originally isolated in the autumn of 1995 from a swine herd insouthern Indiana experiencing a severe PRRS outbreak. This farm had noprevious history of PRRS problems or PRRS vaccination. The P129 isolatewas more virulent than several other field isolates from the same timeperiod and geographic area, in that it produced more severe and moreconsistent respiratory disease in young pigs. The virus was initiallyisolated on primary porcine alveolar macrophage (the natural host cell),and subsequently passaged on MARC-145 cells (Kim et al., 1993). Genesencoding structural proteins of P129 were found to be homologous tocorresponding known North American PRRS gene sequences.

The MARC-145 cell line that was used to propagate PRRS viruses is aclone of the MA-104 Rhesus Macaque Monkey Kidney cell line. The MARC-145cells were obtained from the National Veterinary Services Laboratories(NVSL, Ames, Iowa) of the USDA. These cells have been tested and foundnegative for mycoplasmas and for common porcine extraneous agents.MARC-145 cells are routinely grown at 37 C in OptiMEM (Life TechnologiesInc.) with 2% fetal bovine serum and antibiotics.

Five biological clones were plaque purified from the P129 virus stock,and these were designated P129A through P129E. Plaque purification wascarried out by infecting monolayers of MARC-145 cells with P129 virus,adding an overlay of OptiMEM containing 1.25% SeaPlaque agarose (FMCBioProducts), 2% fetal bovine serum, and antibiotics. Plaques wereclearly visible following incubation for 7 days, when 5 well-isolatedplaques were picked and passaged onto fresh MARC-145 monolayers. Whencytopathic effect (virus induced cell death) became apparent the progenyvirus from each of these cultures was subjected to another round ofplaque purification. One well-isolated plaque from each of the fiveclones was picked and expanded to produce large stocks. The 5 cloneswere tested for virulence in young pigs, either individually (clones Aand E) or in combination (clones B-D, or clones A-E). In all cases, theplaque purified virus replicated well in pigs and caused clinicaldisease. The severity of clinical symptoms was less than that caused bythe uncloned P129 virus, even when all five clones were used together.P129A was chosen for sequencing, and was used in subsequent molecularmanipulations.

Determination of the genome sequence of P129A: Plaque purified virusP129A was used for sequence determination after 10 serial passages fromthe pig (including two plaque purifications and one subsequent passage).SEQ ID NO:1 shows the cDNA sequence corresponding to the P129A RNAgenome. The genome is 15,395 nucleotides in length (excluding thepolyadenosine tail), begins with ATGACGTA, and ends with CCGCAATT. Atypical polyadenosine tail of 55 residues is also provided in SEQ IDNO:1.

For the structural genes of P129A (ORFs 2 through 7), which comprise the3′ 20% of the genome, various PCR primers were chosen based on severalpartial cDNA sequences of other North American PRRS virus isolatesavailable in the public DNA sequence database GenBank (for examplePRU00153). Purified viral RNA was reverse transcribed into cDNA usingreverse transcriptase and random hexamer primers. This cDNA was thenused in PCR with gene-specific primers. PCR products were excised fromgels and T/A cloned into plasmid pCR2.1 (Invitrogen). For each primerpair, multiple plasmids (from independent PCR reactions) were DNAsequenced. Sequences were assembled using the Seqman program from theLasergene package (DNASTAR, Inc). This permitted completing the sequenceof positions 11,992 through 15,347 of the P129A genome.

Also in the GenBank database are a series of short sequences(approximately 218 nucleotides total) which comprise a portion of theORF 1b gene of several isolates of PRRS virus. One of these (PPSSEQB)was used to design PCR primers (forward 5′-ACAGTTTGGTGATCTATG-3′ (SEQ IDNO:10), corresponding to positions 9063-9080; reverse5′-CAGATTCAGATGTTCAA-3′ (SEQ ID NO:11), corresponding to positions9252-9268). These amplified a 206 nucleotide fragments, which includes171 nucleotides of new sequence from the P129A ORF1b gene, correspondsto positions 9081 to 9251. A new forward primer was designed within thisregion (5′-ACCTCGTGCTGTATGCCGAATCTC-3′ (SEQ ID NO:12), positions9201-9224), and a matching primer was designed within ORF1b immediatelyupstream of ORF2 (5′-TCAGGCCTAAAGTTGGTTCAATGA-3′ (SEQ ID NO:13),positions 12,027-12,050). These primers were used in RT-PCR to amplify a2850 nucleotide fragment of ORF1b, corresponding to positions9201-12,050 of the P129A genome.

During RT-PCR amplification of ORF5 of another North American fieldisolate of PRRS virus, a minor band was seen which was smaller than theexpected size. This was sequenced and found to have limited homologywith ORF1a of Lelystad virus (resulting from false priming). New primerswithin this region were chosen to amplify P129A (forward5′-GATGACTGGGCTACTGACGAGGAT-3′ (SEQ ID NO:14), corresponding topositions 1587-1610; reverse 5′-AGAGCGGCTGGGATGACACTG-3′ (SEQ ID NO:15),corresponding to positions 1877-1897). In addition to the product of 311nucleotides (266 nucleotides of new P129A sequence between the primerscorresponding to positions 1611-1876), a larger minor PCR product of 701nucleotides was cloned and sequenced (656 nucleotides of new P129Asequence between the primers corresponding to positions 1611-2264). Thelarger band results from false priming of the reverse primer atpositions 2265-2269.

The extreme 5′ end of the genome of P129A was determined by 5′ RACE(rapid amplification of cDNA ends) using a commercially available kit(Life Technologies Inc). Two nested reverse primers were chosen fromwithin the known ORF1a sequence (“RACE2” 5′-CCGGGGAAGCCAGACGATTGAA-3′(SEQ ID NO:16), positions 1917-1938; and “RACE3”5′-AGGGGGAGCAAAGAAGGGGTCATC-3′ (SEC) ID NO:17), positions 1733-1756).RACE2 was used to prime cDNA synthesis, while RACE3 was used in PCR. Theresulting PCR products were cloned and sequenced. The two longestproducts ended at precisely the same base (position 1 in SEQ ID NO:1).

The large gap between known sequence in ORF1a and ORF1b was bridgedusing long RT-PCR. Two new primers were used (forward5′-AGCACGCTCTGGTGCAACTG-3′ (SEQ ID NO:18), positions 1361-1380; reverse5′-GCCGCGGCGTAGTATTCAG-3′ (SEQ ID NO:19), positions 9420-9438). Theresulting 8078 nucleotide RT-PCR product was cloned and sequenced.

The extreme 3′ end of the genome of P129A was determined by ligating the3′ and 5′ ends of the viral RNA together and using RT-PCR to amplify thejunction fragment. The resulting junction fragments were cloned andsequenced. Briefly, RNA extracted from pelleted virions was treated withtobacco acid pyrophosphatase (to remove 5′ cap structures), thenself-ligated with T4 RNA ligase (both from Epicentre Technologies). Theprimers used were 5′-CGCGTCACAGCATCACCCTCAG-3′ (SEQ ID NO:20) (forward,positions 15,218-15,239) and either 5′-CGGTAGGTTGGTTAACACATGAGTT-3′ (SEQID NO:21) (reverse, positions 656-680) or5′-TGGCTCTTCGGGCCTATAAAATA-3′(SEQ ID NO:22) (reverse, positions337-359). All of the resulting clones were truncated at the 5′ end ofthe genome (the most complete came to within 57 nucleotides of theactual 5′ end, as revealed by 5′ RACE), however two of these clonescontained the complete 3′ end of the genome including the polyadenosinetail (42 and 55 adenosine residues in length). This completed thesequencing of the cDNA 15,450 base genome of PRRS isolate P129A,including polyA tail, as shown in SEQ ID NO:1.

Creation of an infectious full-length cDNA clone of P129A: A full-lengthinfectious cDNA clone of P129A, designated pT7P129A, was assembled fromfour overlapping cloned RT-PCR products. The four RT-PCR products werefirst T/A cloned into plasmid pCR2.1 (Invitrogen) and transfected intoEscherichia coli strain DH5-alpha. Bacterial colonies were screened, andthose which contained inserts of the expected sizes in the “T7 to M13”orientation were chosen for sequencing and further manipulation. Allfour cloned RT-PCR products contained one or more non-silent mutations(deviations from the consensus nucleotide sequence for P129A of SEQ IDNO:1 which would result in a change in amino acid sequence of theencoded ORFs). These non-silent mutations (save one at position 12,622in ORF 2) were repaired by subcloning fragments from other cloned RT-PCRproducts. The four repaired subgenomic clones were assembled into afull-length clone in a stepwise manner, using available restrictionsites (see FIG. 1). The 5′ and 3′ ends of the cDNA corresponding to theP129A genome in pT7P129A were modified by the addition of a T7 promoterand appropriate restriction endonuclease sites. The construction ofpT7P129A is described in further detail in the following paragraphs:

The 5′ end of the genome (positions 1-1756), generated by 5′-RACE andcloned into pCR2.1 as described above, was modified to include a 17promoter immediately upstream of the cDNA corresponding to the P129Agenome and a PacI site for future cloning. A 3-way ligation wasperformed using the 1216 by DsaI-BseRI fragment of this plasmid(containing bases 27-1242 of P129A), the 4407 by BseRI-XbaI fragment ofthe same plasmid (containing bases 1243-1756 of P129A and the entireplasmid vector up to the XbaI site), and the following syntheticdouble-stranded adapter (SEQ ID NO: 23, first below and SEQ ID NO: 24,second below):

5′-CTAGATTAATTAATACGACTCACTATAGGGATGACGTATAGGTGTTGGCTCTATGC-3′3′-TAATTAATTATGCTGAGTGATATCCCTACTGCATATCCACAACCGAGATACGGTGC-5′XbaI           T7 promoter                              DsaI          PacI                 P129A genome

The predicted transcript from the T7 promoter includes a single “G”residue from the promoter immediately upstream of the first “A” of theviral genome. A non-silent mutation at position 1230 (A to G) wasrepaired by replacing the 906 by AatII-SacII fragment (bases 740-1645)with the same fragment from another clone. This plasmid was designated“pT7R3A-2”.

The 8078 nucleotide PCR product described above was used to cover bases1361-9438 of the P129A genome. A 58 by deletion (positions 2535-2592)and 7 non-silent point mutations were corrected by subcloning fragmentsfrom other cloned RT-PCR products, yielding plasmid “pGAP10B-4”. The7837 by Bsu36I-SpeI fragment from this plasmid was ligated to the 5482by Bsu36I-SpeI fragment from pT7R3A-2. The resulting plasmid “pT7RG”contains the first 9438 bases of the P129A genome behind the T7promoter.

The 2850 nucleotide fragment of ORF1b described above (genome positions9201-12,050) was corrected to repair non-silent mutations and designated“p1B3A-2”. The 2682 by NdeI-SpeI fragment of this plasmid was ligated tothe 13,249 by NdeI-SpeI fragment of pT7RG to yield plasmid “pT71A1B”,which contains the first 12,050 bases of the P129A genome.

The fourth and final fragment of the P129A genome was derived by RT-PCRof ORFs 2 through 7, including the 3′ non-translated region and aportion of the polyA tail. The forward primer was5′-ACTCAGTCTAAGTGCTGGAAAGTTATG-3′ (SEC) ID NO:25) (positions11,504-11,530) and the reverse primer was5′-GGGATTTAAATATGCATTTTTTTTTTTTTTT TTTTTTAATTGCGGCCGCATGGTTCTCG-3′ (SEQID NO:26). The reverse primer contains the last 22 bases of the P129Agenome (positions 15,374-15,395), a polyA tail of 21 bases, an NsiI site(ATGCAT) and a SwaI site (ATTTAAAT). Non-silent point mutations and asingle base deletion were repaired by subcloning fragments from otherclones. An additional non-silent point mutation at position 12,622 (A toG) was inadvertently introduced at this stage. This results in a changefrom glutamine to arginine near the C-terminus of the ORF2 protein(amino acid residue 189 of the 256 amino acids in ORF2, which does notaffect the overlapping ORF 3). This mutation had no apparent influenceon viral growth, in cell culture or in pigs, and was not repaired. Thismutation served as a genetic marker to distinguish virus derived fromthe cDNA clone from possible contamination with parental P129A or otherPRRS viruses. The plasmid was designated “p2_(—)7D-4”. The structuralgenes of P129A were added to the rest of the genome by ligating the 3678by Eco47III-SpeI fragment of p2_(—)7D-4 to the 15,635 by Eco47III-SpeIfragment of pT71A1B.

This yields the final construct “pT7P129A”, which comprises cDNAcorresponding almost identically to the entire genome of P129A (however,with only a 21 base polyA tail, as opposed to 55 base polyA tail) behinda T7 promoter, cloned into the pCR2.1 vector between unique restrictionenzyme sites (PacI and SwaI). The total length of pTP7129A is 19,313 bp,and it is stable in E. coli strain DH5-alpha. pT7P129A contains an A toG non-silent point mutation at position 12,622 that results in anarginine at position 189 of ORF2 rather than a glutamine (as is encodedby SEQ ID NO:1) and a silent C to T mutation at position 1559. Neitherof these mutations affected viral growth under the conditions examined,both in cell culture and in pigs. For example, pT7P129A was used for invitro transcription and the resulting RNA transcripts produced liveNorth American PRRS virus when transfected into MARC-145 cells, thusdemonstrating that this full-length clone is infectious.

In vitro transcription and transfection of RNA transcripts: In plasmidpT7P129A there are two T7 promoters in tandem upstream of the viralgenome. One of these is positioned immediately upstream of the viralgenome and was built into the PCR primer as described above. The otheris present in the pCR2.1 cloning vector and is located outside of themultiple cloning site (initiating transcription 44 bases upstream of theviral genome). PacI was used to cut between these T7 promoters prior toin vitro transcription to generate a transcript that is closer toauthentic viral RNA (a single extra G immediately upstream the viralgenome, as opposed to 44 extra bases from the distal T7 promoter). Inaddition, pT7P129A was cut with SwaI prior to in vitro transcription.The resulting run-off transcripts include a 21 base long polyA tail andnine non-PRRS nucleotides, including an NM site (which was not used tolinearize the plasmid, since the site also occurs once in the viralgenome). The digested plasmid was purified by phenol extraction andethanol precipitation prior to use.

A commercial kit (T7 Cap-Scribe, Boehringer Mannheim) was used for invitro transcription. The DNA pellet from above, containing about 0.6 μgof PacI/SwaI digested pT7P129A, was resuspended in 20 μl of T7Cap-Scribe buffer/T7 polymerase and incubated at 37° C. for 30 minutes.A portion of the reaction was analyzed by agarose gel electrophoresisand shown to contain full-length RNA transcripts in addition to theexpected DNA bands of 15,445 by and 3868 bp. The in vitro transcriptionreaction was used fresh, immediately following incubation, withoutpurification. Freshly confluent monolayers of MARC-145 cells were washedonce in OptiMEM (without serum), and covered with 1 ml per 35 mm well ofOptiMEM (without serum) containing 500 μg/ml DEAE dextran (molecularweight approx. 500,000, Pharmacia Biotech). In vitro transcriptionreaction (15 μl) was added immediately. After 1 hour at 37° C., thetransfection mixture was removed, monolayers were washed once with PBSand overlaid with 1.25% SeaPlaque agarose (FMC corporation) in OptiMEMwith 2% fetal bovine serum and antibiotics. After 5 days at 37° C., asingle plaque was visible. This virus was designated “rP129A-1” and wasexpanded on MARC-145 cells and characterized in cell culture and inpigs. Subsequent transfections of in vitro transcribed RNA frompT7P129A, using both DEAE dextran and electroporation, have yielded manyadditional plaques.

Characterization of recombinant virus rP129A-1: There are no apparentdifferences in growth kinetics, yield, or plaque morphology betweencDNA-derived recombinant virus rP129A-1 and its non-recombinant parentP129A. As discussed above, there are two differences in nucleotidesequence between the coding sequence of pT7P129A and the consensussequence of P129A (shown in SEQ ID NO:1). Firstly, at position 1559pT7P129A contains a T, whereas P129A contains a C (this is a silentmutation). Secondly, at position 12,622 pT7P129A contains a G, whereasP129A contains an A (this is the glutamine to arginine change in ORF2described above). In order to rule out the possibility that rP129A-1 isactually a non-recombinant PRRS virus contaminant, RT-PCR and sequencingwere performed on the regions surrounding these two differences. In thecase of both genetic markers, rP129A-1 was identical to plasmid pT7P129Aand different from parental virus P129A, thus confirming that rP129A-1is derived from the infectious cDNA clone.

Characterization of recombinant virus rP129A-1 in pigs: The cDNA-derivedvirus rP129A-1 was compared to its non-recombinant parent P129A for itsability to infect and cause clinical disease in young pigs. Three groupsof 10 pigs each from a PRRS-negative herd were infected at 4 weeks ofage with either P129A, rP129A-1, or mock-infected with cell culturemedium. Clinical signs, rectal temperatures, and body weights weremonitored. Blood was collected on days 0, 2, 6, 10, and 13post-infection for determination of serum viremia (by plaque assay onMARC-145 cells, FIG. 2) and serum antibody (by ELISA using HerdChek PRRSfrom IDEXX, FIG. 3). Gross and microscopic lesions of the lung wereobserved upon necropsy. There were no significant differences betweenthe two virus-infected groups, indicating that rP129A-1 replicates inpigs and causes clinical disease which is quantitatively andqualitatively similar to its non-recombinant parent virus.

Example II Deletion of ORF7 (Nucleocapsid Gene) from the North AmericanPRRS Virus; Preparation of a Negatively-Marked, Replication-DefectiveVaccine Thereby

The viral nucleocapsid gene (ORF7) was partially deleted from aninfectious cDNA clone of the PRRS virus of the present invention. Theresulting recombinant modified PRRS virus would be expected to bereplication-defective in pigs. This recombinant modified PRRS virus canbe used as a vaccine to induce an immune response to the other PRRSvirus proteins without the risks of clinical disease, spread tonon-vaccinated animals, or reversion to virulence associated withattenuated live vaccines. In addition to being very safe, such a vaccinevirus would also be “negatively marked”, in the sense that it wouldallow exposure to field isolates of PRRS virus to be determinedserologically, even in the presence of antibody to the vaccine virus.Antibodies to the ORF7 protein are commonly found in the sera of PRRSvirus-infected pigs, whereas pigs vaccinated with an ORF7-deleted PRRSVwould lack antibodies to the ORF7 protein.

Deletion of ORF7 from an infectious clone was accomplished as follows:Plasmid p2_(—)7D-4 (see FIG. 1) was used as template in PCR to amplifythe 5′ and 3′ flanking regions upstream and downstream of ORF7. Theupstream flank forward primer 5′-ATTAGATCTTGCCACCATGGTGGGGAAATGCTTGAC-3′ (SEQ ID NO:27) (which binds to genomepositions 13,776-13,791 near the beginning of ORFs and containsadditional restriction sites which are irrelevant to the currentcloning) and the upstream flank reverse primer 5′-CTTTACGCGTTTGCTTAAGTTATTTGGCGTATTTGACAAGGTTTAC-3′ (SEQ ID NO:28) (which binds togenome positions 14,857-14,902 at the junction of ORFs 6 and 7)amplified a fragment of 1147 bp. The reverse primer introduced MluI andAflII sites and a single base change at position 14,874, destroying theATG start codon for ORF7 without altering the tyrosine encoded in theoverlapping ORF6. For the downstream flank, the forward primer 5′-CAACACGCGTCAGCAAAAGAAAAAGAAGGGG-3′ (SEC) ID NO:29) (positions 14,884-14,914near the 5′ end of ORF7, introduced an MluI site) and reverse primer5′-GCGCGTTGGCCGATTC ATTA-3′ (SEQ ID NO:30) (downstream of the viralgenome in the pCR2.1 plasmid) amplified a 462 by fragment. A 3-wayligation was performed, using the 611 by BstEII-MluI fragment of theupstream flank PCR product, the 575 by MluI-SpeI fragment of thedownstream flank PCR product, and the 6653 by BstEII-SpeI fragment fromplasmid p2_(—)7D-4 (all fragments were gel purified followingdigestion). The resulting plasmid p2_(—)7Ddelta7₊7023 was deleted in thefirst seven amino acids of ORF7, and lacks a functional ATG start codon.Two new restriction sites which are absent in both the viral genome andthe plasmid backbone, AflII and MluI, have been inserted to facilitatedirectional cloning of foreign genes into the space previously occupiedby the 5′ end of ORF7.

The changes made in p2_(—)7Ddelta7+7023 were incorporated into afull-length genomic clone by ligating the 3683 by Eco47III-SpeI fragmentof p2_(—)7Ddelta7₊7023 with the 15,214 by Eco47III-SpeI fragment ofpCMV-S-p129. The resulting plasmid pCMV-S-p129delta7₊7023 was used totransfect cells.

Since nucleocapsid is essential for viral growth, it is necessary toprovide this protein in order to allow generation and replication of anORF7-deficient PRRS virus. This can be accomplished using a helper virusor a complementing cell line, for example. ORF7-expressing MARC-145 celllines can be created by stably transfecting cells with a plasmidcontaining both the ORF7 gene from P129A and the neomycin resistancegene. After selecting for neomycin resistance using the antibiotic G418,single-cell colonies can then be expanded and characterized. ClonalMARC-145-derived cell lines that are positive for ORF7 expression byboth immunofluorescence and RT-PCR can be transfected with RNA frompT7P129delta7 in order to generate ORF7-deficient P129 virus.

Similar strategies can be used to generate PRRS viruses deficient inother structural genes (ORFs 2, 3, 4, 5, or 6), or deficient in all orportions of non-structural genes 1a and 1b. In addition, multipledeletions can be engineered into a single PRRS virus, and these can begrown in complementing cells which provide all necessary functions. Suchgene-deficient PRRS viruses are likely to be either partially orcompletely attenuated in pigs, making them useful as vaccines againstPRRS. They can also be used to distinguish vaccinated animals fromanimals infected with a wild-type PRRS virus as discussed above and/oras vectors for vaccinating animals with epitopes of other porcinepathogens (see Example III, below).

Example III Insertion of Heterologous Genes into the North American PRRSVirus Genome; use of PRRS Virus as a Vector, and a Positively-MarkedNorth American PRRS Virus

In Example II, above, AflII and MluI restriction enzyme sites wereinserted into the region formerly occupied by the 5′ end of ORF7. Thesesites are absent in the P129A genome and in the pCR2.1 and pCMVplasmids, and can be used in the directional cloning of foreign(heterologous) genes into the viral genome for expression. Potentialleader-junction sites for transcription of the ORF7 subgenomic RNA atpositions 14,744-14,749 (ATAACC) and 14,858-14,863 (TAAACC) are notaffected by deletion of the ORF7 coding sequence, and can function intranscription of a foreign gene. Foreign (heterologous) genes caninclude genes from other PRRS virus isolates or genotypes, and/or genesfrom other non-PRRS pathogens, either pathogens that infect swine orpathogens that infect mammals other than swine, or avians.

In addition, these foreign genes (or portions thereof) can provideantigenic epitopes which are not normally found in swine. Such epitopescan be used to “positively mark” a vaccine, so that successfulvaccination can be monitored serologically, even in the presence ofantibody to field or conventional vaccine strains of PRRS virus. Apositive marker needs not be a separate expression cassette. Anantigenic epitope can be fused to a structural gene of the PRRS virus.For example, the upstream flank reverse primer described in Example I,above, can be extended in such a way as to add a carboxyl-terminalfusion of a non-PRRS virus antigenic epitope to the ORF6 membraneprotein. The presence of antibody to this epitope in swine indicatessuccessful vaccination.

Example IV Cellular Expression of a PRRS Virus by Direct Transfection ofcDNA into Cells

The eukaryotic expression vector pCMV-MC1 (SEQ ID NO:31) was derivedfrom the commercially available plasmid pCMVbeta (Clontech) by replacingthe LacZ coding sequence between two Not I sites with a linkercontaining Not I, EcoR V, Avr II, Bgl II, Cla I, Kpn I, Pac I, Nhe I,Swa I, Sma I, Spe I and Not I sites. Modification of the human CMVimmediate early promoter was accomplished by substituting the sequencebetween the Sac I and the second Not I sites of pCMV-MC1 with asynthetic linker (shown below). The linker contains a half site for SacI following by Pac I, Spe I and a half site for Not I. After annealingthe two single stranded oligonucletides, the linker was cloned intopCMV-MC1 between the Sac I and Not I sites, and a selected clone wasdesignated pCMV-S1. The Spe I site of pCMV-S1 could not be cut, possiblydue to a mistake in the oligo sequence. Therefore, the fragment betweenPac I and Hind III in pCMV-S1 was replaced with Pac I (at position877)-Hind III (at position 1162) fragment from pCMV-MC1. Thus, a Spe Isite was regained. This final construct (pCMV-S) was used to clone thefull length P129 genome.

Linker sequence (SEQ ID NO: 32, first below and SEQ ID NO: 33, secondbelow):

5′ CGTTAATTAAACCGACTAGTGC 3′ 3′ TCGAGCAATTAATTTGGCTGATCACGCCGG 5′           Pac I       Spe I         Sac I                     Not I

The sequence immediately upstream of the 5′ end of the P129 genome wasmodified to contain proper spacing and a convenient restriction enzymesite (Pac I). This was done by designing appropriate PCR primers (SEQ IDNO:34 and SEQ ID NO:35) for amplification from pT7P129. After digestionwith Pac I and Aat II, this PCR fragment was subcloned into the Pac Iand Aat II sites of pT7RG (FIG. 1). The resulting plasmid was designatedpT7RG-deltaT7.

The final construction was completed by subcloning the viral sequencesfrom pT7RG-deltaT7 at the Pac I and Nde I sites into pT7P129, creatingpT7P129-deltaT7. The full length P129 genome was digested frompT7P129-deltaT7 at Pac I and Spe I and transferred into pCMV-S at thePac I and Spe I sites. This constructed was named pCMV-S-P129.

The sequence of the region of modification between the CMV promoter TATAbox and the 5′ end of the P129 sequence in pCMV-S-P129 is shown in SEQID NO:36 and schematically presented below:

   TATATAAGCAGAGCTCGTTAATTAAACCGTCATGACGTATAGGTGTTGGC 5′TATA box  Sac I  Pac I         Start  of  P129  3′

To test the use of the CMV promoter to initiate PRRS virus infection incells, pCMV-S—P129 plasmid DNA (0.5 or 1.0 μg) was transfected intoMARC-145 cells by lipofection using Lipofectamine™ (Life TechnologiesInc.). PRRS virus specific cytopathic effect was observed aftertransfection and the presence of PRRS virus antigen was determined bythe immunofluorescent antibody test.

PRRS virus was generated efficiently from pCMV-S-P129, and the progenyvirus can be passaged on MARC-145 cells. This demonstrates that a PRRSvirus infection can be initiated directly from a plasmid cDNA encoding aPRRS virus, without an in vitro transcription step. Furthermore,pCMV-S-P129 generated a greater amount of progeny virus compared toplasmids wherein the 3′ end of the pCMV promoter was not immediately infront of the start of the sequence encoding the North American PRRSvirus.

Example V Deletion of ORF4 from the North American PRRS Virus;Preparation of a Replication-Defective Vaccine Thereby

A portion of the gene for ORF4, which encodes a membrane glycoprotein,was deleted from an infectious cDNA clone of the PRRS virus of thepresent invention. The resulting recombinant modified PRRS virus isexpected to be replication-defective in pigs and to induce an immuneresponse to the other PRRS virus proteins without the risks of clinicaldisease, spread to non-vaccinated animals, or reversion to virulenceassociated with attenuated live vaccines.

Deletion of ORF4 from an infectious clone was accomplished as follows.Plasmid p2_(—)7D-4 (see FIG. 1) was used as template in PCR to amplifythe 5′ and 3′ flanking regions upstream and downstream of ORF4. Theupstream flank forward primer was 5′-AGGTCGACGGCGGCAATTGGTTTCACCTAGAGTGGCTGCGTCCCTTCT-3′ (SEQ ID NO:37). This primerbinds to genome positions 13194-13241, near the beginning of ORF4, andintroduces a mutation at position 13225 which destroys the ATG startcodon of ORF4 without altering the overlapping amino acid sequence ofORF3. The upstream flank reverse primer was5′-TCTTAAGCATTGGCTGTGATGGTGATATAC-3′ (SEQ ID NO:38). This primer bindsto genome positions 13455-13477 within the ORF4 coding region,downstream of ORF3, and introduces an AflII site. For the downstreamflanking region, the forward primer was 5′-CTTCTTAAGTCCACGCGTTTTCTTCTTGCCTTTTCTATGCTTCT-3′ (SEQ ID NO:39). This primerbinds to genome positions 13520-13545 in the middle of ORF4, andintroduces AflII and MluI sites for directional cloning of foreigngenes. The reverse primer was 5′-TGCCCGGTCCCTT GCCTCT3′ (SEC) ID NO:40).This primer binds to genome positions 14981-14999 in the ORF7 codingsequence. A three-way ligation was performed using the SalI-AflIIfragment of the upstream flank PCR product, the AflII-BstEII fragment ofthe downstream flank PCR product, and the SalI-BstEII fragment fromplasmid p2_(—)7D-4. All fragments were gel-purified following digestion.The resulting plasmid p2_(—)7D-4-delta4N has 42 bases of the centralportion of ORF4 deleted and replaced with a 15 base artificial cloningsite. The cloning site contains two restriction sites (AflII and MluI)that are absent from both the viral genome and the plasmid backbone.These can be used to facilitate directional cloning of foreign genesinto the space previously occupied by ORF4. The cloning site alsocontains a stop codon (TAA) that is in frame with ORF4 and furtherassures that functional ORF4 protein is not produced.

It was found that a more extensive deletion of the ORF4 coding sequencecan be made without interfering with expression of the downstream ORF5envelope gene. In this case a shorter downstream flanking region wasamplified by PCR using the same template and reverse primer, and usingforward primer 5′-GTTTACGCGTCGCTCCTTGGTGGTCG-3′ (SEQ ID NO:41). Thisprimer binds to genome positions 13654-13669 near the 3′ end of ORF4,and contains an NM site. Two-way ligation between the AflII-BstEIIfragment of the downstream flank PCR product and the AflII-BstEIIfragment from plasmid p2_(—)7D-4-delta4N yielded the new plasmidp2_(—)7D-4-delta4NS. This plasmid has 176 bases of the ORF4 codingsequence deleted and replaced with the 15 base cloning site.

The changes made in p2_(—)7D-4-delta4N and p2_(—)7Ddelta4NS wereincorporated into the full-length genomic clone by replacing theBsrGI-SpeI fragment from pCMV-S-P129 with the modified BsrGI-SpeIfragments from p2_(—)7D-4-delta4N and p2_(—)7D-4-delta4NS. The resultingplasmids pCMV-S-P129delta4N and pCMV-S-P129delta4NS were used totransfect cells.

In contrast to pCMV-S-P129, transfection of MARC-145 cells with plasmidspCMV-S—P129delta4N or pCMV-S-P129delta4NS did not result in viralplaques or fluorescent foci. Individual transfected cells can be seen tobe producing the ORF7 nucleocapsid protein, suggesting that the ORF4gene product is not required for RNA replication or expression of viralgenes, but is essential for release of infectious progeny virus. Sincedeletion of ORF4 is lethal to virus replication, it is necessary toprovide this protein. This can be accomplished by using a complementingcell line. We created ORF4-expressing MARC-145 cell lines by stabletransfecting cells with a plasmid containing both ORF4 and the neomycinresistance gene. After selection for neomycin resistance using theantibiotic G418, single-cell colonies were expanded and characterized.After transfection with pCMV-S-P129delta4NS, three ORF4-expressing cellclones yielded live virus that can be propagated in these cells but notin MARC-145 cells. One of these, MARC400E9, was further characterized.Immunofluorescent staining for viral nucleocapsid in MARC400E9 cellstransfected with plasmid pCMV-S-P129delta4NS was positive.

Virus derived by transfecting MARC400E9 cells with thepCMV-S-P129delta4NS plasmid (designated P129delta4NS) was amplified bypassaging several times on MARC400E9 cells and was used to vaccinatepigs against a virulent PRRSV challenge. Live virus was formulated withone of three different types of adjuvant or used without adjuvant, andwas delivered in two doses by one of two vaccination routes (intranasalfollowed by intramuscular, or intramuscular followed by a secondintramuscular dose).

At approximately three weeks (Day 0) and five weeks (Day 14) of age,seronegative conventional pigs were vaccinated according to the Table 2,below. The pigs in Group J were considered sentinel pigs and werenecropsied approximately one week prior to challenge to evaluate thehealth status of all pigs.

TABLE 2 Test groups selected for vaccination derived from P129delta4NSGroup Vaccine N Route* Adjuvant A P129delta4NS 10 IN/IM None BP129delta4NS 10 IM/IM None C P129delta4NS 10 IN/IM rmLT** D P129delta4NS10 IM/IM rmLT E P129delta4NS 10 IN/IM QuilA/Cholesterol F P129delta4NS10 IM/IM QuilA/Cholesterol G P129delta4NS 10 IN/IM Amphigen HP129delta4NS 10 IM/IM Amphigen I PBS only 10 IM/IM None J None 10 NA NA*IN—intranasal; IM—intramuscular. **recombinant mutant heat-labileenterotoxin from E. coli.

The first and second intramuscular vaccinations were administered in theleft and right sides of the neck, respectively. On Days 0, 13, 21 and28, blood samples were collected for serology (IDEXX HerdChek PRRSELISA). At approximately three weeks following the second vaccination(Day 35), all pigs were challenged with 1 mL of parental P129 virusinstilled drop-wise in each nostril (a total of 2 mL/pig or 1.2×10⁵pfu). On Days 35, 38, 42 and 45, blood samples were collected from allpigs for serology and for PRRSV titration (by TCID50 assay). On Days 45and 46, pigs were euthanized and necropsied. Lungs were removed from thechest cavity, examined for PRRS lesions, and photographed. Necropsieswere performed at the Animal Disease Research and Diagnostic Laboratory(ADRDL) at South Dakota State University, Brookings, S. Dak.

The serological results showed induction of anti-PRRSV antibody inseveral of the vaccinated groups prior to challenge, and priming of thehumoral immune system for a rapid post-challenge antibody response inall vaccinated groups. Immediately before challenge (Day 35), theaverage S/P ratio (Sample/Positive control ratio) value in theunvaccinated controls (group I) was 0.003. All eight of the vaccinatedgroups had higher values, ranging from 0.005 (group B) up to 0.512(group H). By one week after challenge (Day 42), the control group wasbeginning to mount an antibody response to the challenge virus (S/Pvalue of 0.171). All eight vaccinated groups had much higher antibodylevels, ranging from 0.858 (group B) to 1.191 (group G). This representsan increase in anti-PRRSV antibodies of approximately 5- to 10-fold as aresult of priming of the humoral immune system.

TABLE 3 Anti-PRRSV antibody levels (S/P ratios) Day-1 Day-13 Day-21Day-28 Day-35 Day-38 Day-42 Day-45 Group A None/IN 0.003 0.008 0.0040.002 0.015 0.102 0.862 2.270 Group B None/IM 0.005 0.015 0.024 0.0220.005 0.105 0.858 2.053 Group C rmLT/IN 0.002 0.030 0.015 0.021 0.0160.108 1.219 2.653 Group D rmLT/IM 0.002 0.042 0.143 0.149 0.138 0.2291.271 2.679 Group E QuiIA/IN 0.015 0.039 0.084 0.048 0.013 0.097 1.2172.299 Group F QuiIA/IM 0.001 0.020 0.104 0.115 0.085 0.109 1.240 2.593Group G Amph/IN 0.002 0.001 0.047 0.051 0.061 0.080 1.919 3.484 Group HAmph/IM 0.022 0.006 0.460 0.531 0.512 0.492 1.601 3.371 Group I PBS only0.008 0.020 0.001 0.001 0.003 0.053 0.171 0.881

Following challenge with virulent PRRSV, serum viremia was determined.At three days post challenge (Day 38) the titer of virus in theunvaccinated control group had reached 2.58 logs. All eight of thevaccinated groups showed reduced viremia, ranging from 2.52 (group E)down to 1.07 logs (group H). This represents more than a 30-foldreduction in virus levels relative to the control group, see Table 4.

TABLE 4 Serum viremia (log 10 TCID50 titers) on day 38 (three days postchallenge) Group A None/IN 2.18 Group B None/IM 1.98 Group C rmLT/IN2.15 Group D rmLT/IM 2.48 Group E QuilA/IN 2.52 Group F QuilA/IM 1.65Group G Amph/IN 1.74 Group H Amph/IM 1.07 Group I PBS only 2.58

At necropsy, all pigs were examined for the presence of macroscopic lunglesions resulting from the PRRS challenge virus. In the unvaccinatedcontrol group, 90% of the animals showed lung lesions. In contrast, alleight of the vaccinated groups showed reduced levels of lung lesions(ranging from 80% in group D down to 22% in group A). Vaccinationreduces damage to the lungs that result from PRRSV infection. See Table5.

TABLE 5 Number of pigs with macroscopic lung lesions/total pigs (percentpositive) Group A None/IN  2/9 (22%) Group B None/IM 7/10 (70%) Group CrmLT/IN 6/10 (60%) Group D rmLT/IM 8/10 (80%) Group E QuilA/IN 5/10(50%) Group F QuilA/IM 4/10 (40%) Group G Amph/IN 7/10 (70%) Group HAmph/IM  5/9 (56%) Group I PBS only 9/10 (90%)

The above swine data demonstrates the process of vaccinating swine withthe recombinant North American PRRS virion containing the ORF 4deletion. The results of the above data illustrate that the P129delta4NSvirus, which is deleted in ORF4, is useful as a vaccine to protect pigsfrom disease caused by the PRRS virus. Efficacy of the vaccine may beincreased by formulation with various adjuvants and by changing theroute of administration.

Example VI Use of Gene-Deleted PRRS Virus as a Vector for Expression ofForeign Genes

In order to determine whether heterologous genes can be expressed from agene-deleted PRRS virus, we inserted a copy of green fluorescent protein(GFP) into the site of ORF4 deletion in plasmid pCMV-S-P129delta4N. TheGFP gene was amplified from a commercially available plasmid vectorusing PCR primers that introduced an AflII site at the 5′ end of thegene and an MI site at the 3′ end of the gene. The resulting plasmidpCMV-S—P129delta4N-GFP was used to transfect MARC-145 and MARC400E9cells. As anticipated, MARC-145 cells did not support replication of theORF4-deleted virus. Single green cells resulting from primarytranfection were seen under the UV scope, indication that GFP was beingexpressed, but the virus did not spread to neighboring cells and no CPEwas observed. In contrast, large green foci were observed in transfectedMARC400E9 cells. Visible plaques formed and merged, destroying themonolayer. These results indicate that foreign genes can be expressedfrom ORF4-deleted PRRS virus, and that increasing the size of the viralgenome by 692 bases (4.5%) does not interfere with packaging of theviral RNA into infectious particles.

Example VII Use of Replication-Competent PRRS Virus as a Vector forExpression of Foreign Genes

In order to determine whether heterologous genes can be expressed from areplication-competent PRRS virus, we inserted a copy of GFP into theregion between ORF1b and ORF2. Since the leader/junction (L/J) sequencefor ORF2 lies within ORF1b, this L/J sequence was used to driveexpression of the GFP gene and a copy of the ORF6 L/J sequence wasinserted downstream from GFP to drive expression of ORF2.

Plasmid p2_(—)7D-4 (see FIG. 1) was used as template in PCR to amplifythe 5′ and 3′ flanking regions upstream and downstream of the insertionsite. The upstream flank forward primer was5′-AACAGAAGAGTTGTCGGGTCCAC-3′ (SEC) ID NO:42). This primer binds togenome positions 11699-11721 in ORF1b. The upstream flank reverse primerwas 5′-GCTTT GACGCGTCCCCACTTAAGTTCAATTCAGGCCTAAAGTTGGTTCA-3′ (SEQ IDNO:43). This primer binds to genome positions 12031-12055 in ORF1b andadds AflII and MluI sites for directional cloning of foreign genesbetween ORF1b and ORF2. The downstream flank forward primer was5′-GCGACGCGTGTTCCGTGGCAACCCCTTTAACCAGAGTTTCAGCGGAACAATGAAATGGGGTCTATACAAAGCCTCTTCGACA-3′ (SEC) ID NO:44). This primerbinds to genome positions 12056-12089 in ORF2 and contains an MluI sitefollowed by the 40 bases that precede the start of ORF6 (containing theORF6 L/J sequence). The downstream flank reverse primer was5′-AACAGAACGGCACGATACACCACAAA-3′ (SEQ ID NO:45). This primer binds togenome positions 13819-13844 in ORF5. A three-way ligation was performedusing the Eco47III-MluI fragment of the upstream flank PCR product, theMluI-BsrGI fragment from the downstream flank PCR product, and theEco47III-Bs/GI fragment from pCMV-S-P129. The resulting plasmidpCMV-S-P129-1bMCS2 contains the entire P129 genome with a cloning siteand an additional L/J site between ORF1b and ORF2. The plasmid producesfunctionally normal virus when transfected into MARC-145 cells.

The GFP gene from a commercially available plasmid was PCR amplifiedusing primers that add an NM site to the 5′ end and an MluI site to the3′ end of the gene. After digestion of the PCR fragment andPCMV-SP129-1bMCS2 with MAI and MluI, the insert was ligated into thevector to yield plasmid pCMV-S-P129-1bGFP2. This plasmid produced greenplaques when transfected into MARC-145 cells. The resulting virus can bepassaged onto MARC-145 cells and continued to produce green plaques whenobserved under the UV scope. Thus, foreign genes may be expressed fromreplication-competent PRRS virus vectors. The P129-1 bGFP2 viruscontains a genome which is 774 bases (5%) longer than that of its P129parent, yet it is packaged normally.

Example VIII Deletion of ORF 2 (Minor Structural Protein) from the NorthAmerican PRRS Virus; Preparation of a Replication-Defective Vaccine

The following example illustrates the preparation of the North AmericanPRRS virions containing the ORF 2 deletion.

The viral minor structural protein encoded by ORF2 is deleted from aninfectious cDNA clone of the PRRS virus of the present invention. Theresulting recombinant modified PRRS virus is replication-defective inpigs and can induce an immune response to the other PRRS virus proteinswithout the risks of clinical disease, spread to non-vaccinated animals,or reversion to virulence associated with attenuated live vaccines.

Deletion of ORF2 from an infectious clone was accomplished as follows.Plasmid p2_(—)7D-4 (see FIG. 1) was used as template in PCR to amplifythe 5′ and 3′ flanking regions upstream and downstream of ORF2. Theupstream flank forward primer was 5′-CAAAGGGC GAAAAACCGTCTATCA-3′ (SEQID NO:46), which binds within the cloning vector. The upstream flankreverse primer was 5′-CCCCACTTAAGTTCAATTCAGGC-3′ (SEQ ID NO:47), whichbinds to genome positions 12045-12067 near the beginning of ORF2. Thedownstream flank forward primer was5′-GCTCCTTAAGAACAACGCGTCGCCATTGAAGCCGAGA-3′ (SEC) ID NO:48), which bindsto genome positions 12492-12528. The downstream flank reverse primer was5′-GCAAGCCTAATAACGAAGCAAATC (SEQ ID NO:49), which binds to genomepositions 14131-14154. A 3-way ligation was performed, using theXhoI-AflII fragment of the 5′ flanking PCR product, the AflII-BsrG1fragment of the 3′ flanking PCR product, and the large XhoI-BsrGIfragment from plasmid p2_(—)7D-4. All fragments were gel purifiedfollowing restriction enzyme digestion. The resulting plasmidp2_(—)7D4delta2 was deleted in the region of ORF2 that does not overlapORF3 or it's 5′ transcriptional regulatory sequences. Two restrictionsites that are absent in both the viral genome and the plasmid backbone,AflII and MluI, have been introduced to facilitate directional cloningof foreign genes into the space previously occupied by ORF2.

The changes made in p2_(—)7D4delta2 were incorporated into a full-lengthgenomic clone by replacing the AfeI-BsrG I fragment from pCMV-S-P129with the modified sequence from p2_(—)7D4delta2. The resulting plasmidpCMV-S-P129delta2 was used for transfections.

Since the deletion of ORF2 is lethal to virus replication, it isnecessary to provide this protein in trans to allow generation ofORF2-deficient PRRS virus. This can be accomplished by using acomplementing cell line. We created ORF2-expressing MARC-145 cell lineswere created by stably transfecting cells with a plasmid containing boththe ORF2 gene from P129A and the neomycin resistance gene. Afterselecting for neomycin resistance using the antibiotic G418, single-cellcolonies were expanded and characterized. After transfecting withpCMV-S-P129delta2, three cell clones yielded live viruses that can bepropagated and passed only in these ORF2-expressing cells. This viruswas designated P129delta2.

Recombinant virus P129delta2 was used to vaccinate groups of 20sero-negative conventional pigs. At three weeks of age (Day 0) and sixweeks of age (Day 20), pigs were vaccinated according to Table 6, below.Controls were mock vaccinated with phosphate buffered saline (PBS).Amphigen adjuvant was not viricidal to the live vaccine virus.

TABLE 6 Various groups of 20 sero-negative conventional pigs vaccinatedwith P129delta2. Treatment Group Route* Adjuvant Titer/Dose (log10TCID₅₀) P129delta2 IM IM/IM 5% Amphigen 5.8 P129delta2 IT IT/IT None 5.7Mock vaccinated (PBS) Controls IT/IT None NA *IM—intramuscular;IT—intratracheal.

At three weeks following second vaccination (Day 42, 9 weeks of age),pigs were challenged with approximately 2.8 mL of virulent P129challenge virus (3.41×10⁵ TCID₅₀/mL) in each nostril, to achieve a totalchallenge volume of 5.6 mL (1.91×10⁶ TCID₅₀). Pigs were bledperiodically for determination of anti-PRRSV antibody (IDEXX HerdChekPRRS ELISA) and serum viremia (TCID₅₀ assay).

TABLE 7 Anti-PRRSV antibody levels (S/P ratios) Day 2 Day 19 Day 28 Day34 Day 42 Day 52 Day 55 Day 63 P129delta2 IM 0.026 0.132 1.265 1.3191.389 1.995 2.322 2.02 P129delta2 IT 0.011 0.004 0.026 0.115 0.036 1.3061.584 1.501 Mock vaccinated 0.001 0.002 0.071 0.003 0.004 0.763 1.3621.395 (PBS) Controls

Two vaccinations with live Amphigen-adjuvanted P129delta2intramuscularly resulted in a marked increase in anti-PRRSV antibody inthe serum prior to challenge, as well as an anamnestic responsefollowing challenge. Two intratracheal vaccinations with unadjuvantedP129delta2 were less effective, but nevertheless resulted in a slightincrease in antibody level prior to challenge and a more rapid responsefollowing challenge, relative to the mock-vaccinated control group.

TABLE 8 Results from two vaccinations with live Amphigen-adjuvantedP129delta2 and control. Serum titer (log 10 TCID₅₀) and number ofpositive pigs following PRRSV challenge Days post-challenge Treatment 05 8 10 14 21 P129delta2 IM Titer 0.0 1.5 0.3 0.0 0.0 0.0 Positive/Total0/19 13/18  4/18  1/18 0/8 0/8 (%) (0%) (72%) (22%)  (6%)  (0%) (0%)P129delta2 IT Titer 0.0 2.3 2.1 1.5 0.5 0.2 Positive/Total 0/20 19/2019/20 17/20  4/10  1/10 (%) (0%) (95%) (95%) (85%) (40%) (10%)  Mockvaccinated Titer 0.0 2.4 2.0 1.4 0.3 0.0 (PBS) Controls Positive/Total0/20 19/19 19/19 16/19 3/9 0/9 (%) (0%) (100%)  (100%)  (84%) (33%) (0%)

Intramuscular vaccination with two doses of live Amphigen-adjuvantedP129delta2 virus resulted in a reduction of the number of viremic pigs(from 100% to 72%), reduction in the duration of viremia (from about 21days to about 14 days), and reduction in the average serum virus load ofnearly one log at 5 days post-infection (the peak of viremia) and morethan one log at later times. Intratracheal vaccination with unadjuvantedP129delta2 showed little or no reduction in viremia relative to themock-vaccinated control group.

Example IX Insertion of Unique MluI and SgrAI Restriction Sites into thePRRS Virus Genome within nsp2

The QuikChange® II XL Site-Directed Mutagenesis kit (Stratagene; LaJolla, Calif.) was used to introduce a unique MluI restriction enzymesite into the PRRS virus genome at nucleotide position 3,219 usingplasmid pCMV-S-P129 as template. Genome position 3,219 lies within thenonstructural protein 2 (nsp2) coding region, which is part of ORF1a(FIG. 5). This site is located within the hypervariable C-terminalportion of nsp2. The sequence of the parental virus beginning at genomeposition 3,219 (ACACGC) is such that changing only two nucleotides(ACGCGT) allows the introduction of an MI site without altering theencoded amino acid sequence. The mutagenic primers used to insert theMluI site (MluI F and MluI R) are shown in Table 1. QuikChange II XLreactions were as recommended by the manufacturer and contained 150picomoles of each primer and 10 ng of plasmid DNA as template. Reactionconditions were as follows: initial denaturation at 95° C. for 60seconds, 18 cycles of 95° C. denaturation for 50 seconds, 60° C.annealing for 50 seconds, and 68° C. extension for 20 minutes,concluding with a 7 minute incubation at 68° C. Successful introductionof the new site was determined based on the ability of the resultingplasmid to be cut with MluI, and confirmed by DNA sequencing. The newplasmid was named pCMV-S-P129-Nsp2-Mlu, and was used as a backbone forthe insertion foreign DNA fragments.

A unique SgrAI restriction enzyme site was inserted into plasmidpCMV-S-P129-Nsp2-Mlu as follows. A 4500 by PCR product (genome positions3201-7700) was amplified using template pCMV-S-P129-Nsp2-Mlu, primersMlu-F and 7700-R (Table 1), and ExTaq™ polymerase (TakaRa Minus Bio;Madison, Wis.) according to the manufacturer's directions. The forwardprimer Mlu-F contains the unique MluI site added in the previous step,and the reverse primer 7700-R contains a unique PmeI site to be used ina subsequent cloning step. Reactions conditions were: initialdenaturation at 95° C. for 120 seconds, 30 cycles of 94° C. denaturationfor 35 seconds, 58° C. annealing for 35 seconds, and 72° C. extensionfor 4.5 minutes. The resulting PCR product was cloned the TOPO PCR XLcloning kit (Invitrogen Corp.; Carlsbad, Calif.). The QuikChange II XLSite-Directed Mutagenesis Kit was used to introduce a unique SgrAI sitebeginning at genome position 3614 according to the manufacturer'sdirections. The original sequence TACCGGTG was changed to CACCGGTGwithout altering the encoded amino acid sequence, using primers SgrAI-Fand SgrAI-R (Table 1). Reactions conditions were: initial denaturationat 95° C. for 60 seconds, 18 cycles of 95° C. denaturation for 50seconds, 60° C. annealing for 50 seconds, and 68° C. extension for 9minutes, followed by a 68° C. polishing reaction for 7 minutes. Aftertransformation, plasmids containing the new SgrAI site were selected byrestriction digestion and sequence analysis. The 4,456 by MluI-PmeIfragment from this plasmid was ligated to the 14,436 by MluI-PmeIfragment of PCMV-S-P129-Nsp2-Mlu to yield pCMV-S-P129-Nsp2-Mlu/SgrA.This plasmid was transfected into MARC-145 cells using Lipofectamine2000 (Invitrogen Corp.; Carlsbad, Calif.) according to themanufacturer's instructions. Three days post-transfection lysates weretitrated by limiting dilution. Infectious virus was recovered fromplasmid pCMV-S-P129-Nsp2-Mlu/SgrA at levels that were similar toplasmids pCMV-S-P129 and pCMV-S-P129-Nsp2-Mlu.

Example X Insertion of Green Fluorescent Protein (GFP) into nsp2

MluI sites were placed on the 5′ and 3′ ends of GFP, amplified by PCRfrom plasmid pEGFP-C3 (Clontech, Mountain View, Calif.), usinggene-specific PCR primers GFP-Mlu-F and GFP-Mlu-R (Table 1). ExTaq™polymerase (TakaRa Mins Bio; Madison, Wis.) was used according to themanufacturer's recommendation. Reactions conditions were: initialdenaturation at 95° C. for 120 seconds, 30 cycles of 94° C. denaturationfor 35 seconds, 56° C. annealing for 35 seconds, and 72° C. extensionfor 60 seconds. The PCR product was digested with MluI and ligated intoMluI-digested pCMV-S-P129-Nsp2-Mlu. DNA sequencing confirmed that GFPhad been inserted in-frame, in the forward orientation, into nsp2. Thenew plasmid was designated pCMV-S-P129-Nsp2-Mlu-GFP.

Plasmids pCMV-S-P129, pCMV-S-P129-Nsp2-Mlu, and pCMV-S-P129-Nsp2-Mlu-GFPwere transfected into the MARC-145 monkey kidney cell line in order togenerate infectious virus. Cell lines were maintained at 37° C., 5% CO₂in modified Eagle's medium (MEM) supplemented with 8% fetal bovineserum, 0.008% Fungizone and 0.01% penicillin/streptomycin. Approximatelyone day prior to transfection, 2×10⁵ cells/well were seeded into a12-well plate. Transfection of infectious cDNA clones was performedusing Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions. At 4 days post-transfection, virus washarvested by subjecting the infected cells to three freeze-thaw cycles.Cell debris was removed by centrifugation at 1000×g for 10 minutes, andvirus-containing culture supernatants collected. Recombinant virusesrescued from plasmids pCMV-S-P129, pCMV-S-P129-Nsp2-Mlu, andpCMV-S-P129-Nsp2-Mlu-GFP were designated P129, P129-Nsp2-Mlu, andP129-Nsp2-Mlu-GFP, respectively.

To determine virus titer, end-point dilutions were performed on MARC-145cells in 96 well plates. Titrations were performed in triplicate andresults presented as TCID₅₀/ml. Cells were fixed at 3 dayspost-transfection and immunostained either directly with thenucleocapsid-specific monoclonal antibody SDOW-17 conjugated to FITC, orindirectly with the nucleocapsid-specific primary monoclonal antibodiesSR30 or JP24 followed by Alexaflour 594-conjugated goat anti-mouse IgGsecondary antibody (Molecular Probes/Invitrogen Corp.; Carlsbad,Calif.). Titers of P129 and P129-Nsp2-Mlu were similar, while the titerof P129-Nsp2-Mlu-GFP was slightly lower. The P129-Nsp2-Mlu-GFP virusexpressed GFP and formed fluorescent green foci. Within the focus ofinfection, cells with perinuclear green fluorescence are seen, due toexpression of the Nsp2-GFP fusion peptide.

For western blot analysis, cells were lysed with 100 ul of 2× Laemmlisample buffer and boiled for 10 min. Proteins were fractionated onSDS-polyacrylamide gels (Bio-Rad; Hercules, Calif.) under reducingconditions. Proteins were transferred onto nitrocellulose membranes andprocessed at room temperature. Membranes were blocked for 1 hr with 5%nonfat dry milk in washing solution (0.2% Tween 20 in PBS). Affinitypurified goat anti-GFP antibody (Rockland Immunochemicals Inc.,Gilbertsville, Pa.) was diluted 1:1000 in blocking buffer and incubatedwith membranes for 1 hr. Membranes were washed three times for 15minutes with TBS-Tween. Rabbit anti-goat horseradishperoxidase-conjugated antibody (Rockland) was diluted 1:1000 in blockingbuffer and incubated with membranes for 1 hr. Membranes were washedthree times for 15 minutes with TBS-Tween. Peroxidase activity wasdetected using the Supersignal West Pico Chemiluminescent substrate(Pierce Chemical Company; Rockford, Ill.). Western blots identified a 70kDa peptide from P129-Nsp2-Mlu-GFP infected cells that reacted withanti-GFP antibody.

Using a similar procedure, the GFP gene was also inserted into nsp2 atthe SgrAI site in the plasmid pCMV-S-P129-Nsp2-Mlu/SgrA. PrimersGFP-SgrA-F and GFP-SgrA-R (Table 1) were used to add SgrAI sites to theends of the GFP gene, using plasmid pEGFP-C3 as template. ExTaq™polymerase (TakaRa Mirus Bio; Madison, Wis.) was used according to themanufacturer's recommendation. Reactions conditions were: initialdenaturation at 95° C. for 120 seconds, 30 cycles of 94° C. denaturationfor 35 seconds, 55° C. annealing for 35 seconds, and 72° C. extensionfor 35 seconds. The PCR product was digested with SgrAI and ligated intoSgrAI-digested pCMV-S-P129-Nsp2-Mlu/SgrA. DNA sequencing confirmed thatGFP had been inserted in-frame, in the forward orientation, into nsp2.The new plasmid was designated pCMV-S-P129-Nsp2-SgrA-GFP. This plasmidproduced infectious virus and fluorescent green foci when transfectedinto MARC-145 cells.

TABLE 9 PCR primers used to Introduce the heterologous genes. Primer(genome SEQ position) Sequence Orientation ID NO MluI-FCTGTCAAGTGTTAGGATCACGCGTCCAAAATACTCAG Forward 50 (3201-3242) CTCAAMluI-R TTGAGCTGAGTATTTTGGACGCGTGATCCTAACACTT Reverse 51 (3201-3242)GACAG GFP-Mlu-F GCCACGCGTGCCACCATGGTGAGCAAGGGCGAG Forward 52 GFP-Mlu-RGCCACGCGTGTTATCTAGATCCGGTGGATCC Reverse 53 7700-RTCAAGCCGCTGGCGGCTAGCAGTTTAAACACTGCTC Reverse 54 (7661-7700) CTTA SgrAI-FCTCGGGAAAATAGAAAACACCGGTGAGATGATCAACC Forward 55 (3597-3637) AGGGSgrAI-R CCCTGGTTGATCATCTCACCGGTGTTTTCTATTTTCC Reverse 56 (3597-3637)CGAG GFP-SgrA-F GCCGCCCACCGGTGAGGTGAGCAAGGGCGAGGAGC Forward 57 TGTTCGFP-SgrA-R GCCGCCCACCGGTGTTGTTATCTAGATCCGGTGGAT Reverse 58 CAll sequences are 5′to 3′. Restriction sites are underlined. Nucleotide positions are based on the sequence of PRRSV strain P129. 

Example XI Construction of PRRS Viruses Having a Deleted Portion of nsp2

The previously made plasmid pCMV-S-P129-Mlu/SgrA (see Example IX) wasused as the backbone plasmid for construction of recombinant PRRSviruses lacking a 132 amino acid region of nsp2, and with either greenfluorescent protein (GFP) or the influenza hemagglutinin (HA) taginserted into the deleted region.

The enhanced GFP gene from a commercially available plasmid (pEGFP-C3,Clontech) was PCR amplified using primers that add MluI and SgrAIrestriction enzyme recognition sites to the 5′ and 3′ ends,respectively. The forward primer was GFP-MLU-F2(5′-GCCACGCGTGTGAGCAAGGGCGAGGAGCTG-3′) SEQ ID NO. 59 and the reverseprimer was GFP-SgrA-R (5′-GCCCACCGGTGTTGTTATCTAGATCCGGTGGATC-3) SEQ IDNO. 58. PCR reactions used ExTaq polymerase (TakaRa Mirus Bio, Madison,Wis.) according to the manufacturer's instructions. Reaction conditionsincluded an initial 120 second denaturation at 95 C, followed by 30cycles of denaturation at 94 C for 35 seconds, annealing at 55 C for 35seconds, and extension at 72 C for 35 seconds. After digestion of thePCR fragment and of pCMV-S-P129-Mlu/SgrA with MluI and SgrAI, the insertwas ligated into the vector to yield plasmid pCMV-S-P129-Mlu-GFP-SgrA.Deletion of the 132 amino acid region and insertion of GFP (257 aminoacids) was confirmed by DNA sequencing. This plasmid produced viablevirus, which in turn produced green fluorescent foci of infection whentransfected into MARC-145 cells and observed under a fluorescencemicroscope. This virus was designated P129-Mlu-GFP-SgrA.

The HA tag is a small peptide epitope (YPYDVPDYA), derived from theinfluenza virus hemagglutinin gene, that reacts with commerciallyavailable monoclonal antibodies. A synthetic adapter was created byheating and slowly annealing two phosphorylated oligonucleotides,designated “HA tag F” (5′-CGCGTATACCCATACGACGTCCCAGACTACGCA-3′) SEQ IDNO. 60 and “HA tag R” (5′-CCGGTGCGTAGTCTGGGACGTCGTATGGGTATA-3′) SEQ IDNO. 61. This adapter, with MluI and SgrAI half-sites, was then ligatinginto pCMV-S-P129-Mlu/SgrA which was previously digested with MluI andSgrAI. The structure of the resulting plasmid, pCMV-S-P129-Mlu-HA-SgrA,was confirmed by DNA sequencing. An in-frame fusion of the HA tagpeptide 9-mer replaces the 132 amino acid deletion in nsp2. The plasmidproduced viable virus when transfected into MARC-145 cells. This viruswas designated P129-Mlu-HA-SgrA.

In a separate experiment, a simple deletion of the same region (withouta simultaneous insertion) was engineered into a different infectiouscDNA clone, using a different methodology. The plasmid pCMV-S-P129-PKcontains a full-length infectious cDNA clone of the P129 isolate of PRRSvirus that was isolated from the serum of an infected pig on primaryporcine alveolar macrophage cells, and subsequently passaged 16 times onthe porcine kidney cell line PK.9. The PK.9 cell line expresses theCD163 PRRS virus receptor, and is fully permissive to PRRSV infection. Aprecise deletion of 396 nucleotides, which encodes the 132 amino acidregion described above, was generated in pCMV-S-P129-PK using theQuikChange II XL Site-Directed Mutagenesis kit (Stratagene; La Jolla,Calif.) and mutagenic primers P129-PK-3199F(5′-CCCTGTCAAGTGTTAAGATCACAGGTGAGATGATCAACC-3′) SEQ ID NO. 62 andP129-PK-3637R (5′-CCCTGGTTGATCATCTCACCTGTGATCTTAACACTTGACAG-3′) SEQ IDNO. 63. The resulting plasmid pCMV-S-P129-PK-dnsp2 contained a deletionof nsp2 amino acids 628-759 (confirmed by sequencing), and producedviable progeny virus when transfected into PK.9 cells. This virus wasdesignated P129-PK-dnsp2.

Example XII Development of an ELISA for the Detection of SerumAntibodies to a 132-Amino Acid Portion of nsp2

The 132 amino acid portion of nsp2 that is deleted in virusesP129-Mlu-GFP-SgrA and P129-Mlu-HA-SgrA is designated P129-nsp2(628-759). This peptide,RPKYSAQAIIDLGGPCSGHLQREKEACLRIMREACDAAKLSDPATQEWLSRMWDRVDMLTWRNTSAYQAFRTLDGRFGFLPKMILETPPPYPCGFVMLPHTPAPSVSAESDLTIGSVATEDIPRILGKIENT) (SEQ ID NO. 64) was expressed as a recombinant peptide inEscherichia coli, using the pHUE system (Catanzariti A M, Soboleva T A,Jans D A, Board P G, Baker R T. 2004. An efficient system for high-levelexpression and easy purification of authentic recombinant proteins.Protein Sci. 13:1331-1339). In this expression system, the peptide ofinterest is cloned and expressed as a histidine-tagged/ubiquitin/PRRSpeptide fusion (25 kDa apparent molecular weight on SDS-PAGE). After aninitial round of purification by nickel ion affinity chromatography, thehistidine-tagged/ubiquitin/PRRS peptide is optionally incubated withhistidine-tagged/deubiquinating enzyme to cleavehistidine-tagged/ubiquitin from the PRRS peptide. In a second round ofnickel ion affinity chromatography, the histidine-tagged/ubiquitin andthe histidine-tagged/deubiquinating enzyme are retained on the nickelion affinity column and the free PRRS peptide is eluted.

The ubiquitin-conjugated P129-nsp2 (628-759) peptide from the firstnickel affinity chromatography step was coated on ELISA plates (0.1ug/ml). Plates were blocked with 10% goat serum. Dilutions of sera frominfected and non-infected pigs were made in PBS with 10% goat serum, andadded to the blocked plates. After incubation and washing, bound porcineantibodies were detected using biotin-labeled goat anti-swine antibody,followed by avidin-peroxidase. Peroxidase activity was detected using achromagenic substrate.

Serum from an uninfected control pig was serially diluted from 1:10 to1:20,480 and applied to the ELISA plates for analysis. Optical density(OD) was below 0.4 in all cases. In contrast, serum from a pig that hadpreviously been infected with the heterologous PRRS virus isolate VR2332about 200 days earlier reacted in a dilution-dependent manner, giving ODvalues in excess of 1.2 at 1:10 and 1:20 dilutions, and dropping belowan OD of 0.4 only after reaching a 1:640 dilution. The VR2332 virus isquite divergent from the P129 isolate, and the 132 amino acid nsp2(628-759) peptide from VR2332(RPKYSAQAIIDSGGPCSGHLQEVKETCLSVMREACDATKLDDPATQEWLSRMWDRVDMLTWRNTSVYQAICTLDGRLKFLPKMILETPPPYPCEFVMMPHTPAPSVGAESDLTIGSVATEDVPRILEKIENV) (SEQ. ID NO. 65) shares only 85.6% identity with its homologthat was used in the ELISA assay. This suggests that antigenic epitopesare shared, and that an ELISA of this sort is likely to be robust enoughto detect infection with most, if not all, type II (“North American”genotype) PRRS viruses. In addition, the ELISA is sensitive enough toshow a strong positive reaction with serum from a pig whose antibodylevels have been waning for a long period of time (200 days postinfection).

Pigs vaccinated with a virus that lacks the nsp2 (628-759) peptide, dueto genetic deletion, are not expected to induce antibodies to thisregion. It is this ability to differentiate vaccinated and naïve(negative) pigs from pigs that have been exposed to field strains of thePRRS virus (positive) that form the basis of the utility of thisinvention. Pigs that test positive can be removed from the herd orplaced in isolation, thus aiding in the management and possibleeradication of the PRRS virus.

Alternatively to the peptides disclosed above, peptides representing thecorresponding region from other PRRS viruses can be employed in adiagnostic assay. Numerous examples of such peptides are provided for bythe sequences disclosed in SEQ ID. NOs 66-115. That is 50 examples ofsimilarly suitable peptides are provided by the polypeptides disclosedin SEQ ID. NOs 66-115 In addition to those examples, fragments of any ofthe peptides described can be used as a reagent. Fragments can be asshort as ten amino acids, capable of forming and being recognized as anepitope by an antibody molecule, or as long as the entire region. Forexample, the peptide can comprise amino acids 1-10 of the respective 132amino acid nsp2 peptide. The peptide can comprise amino acids 2-11 ofthe peptide. The peptide can comprise amino acids 3-12 of the peptide.Peptides are selected in this fashion by progressively “walking” downthe amino acid sequence of the nsp2 fragment, all of the way through toamino acid 132, generating 123 possible 10-mer peptides.

In addition to 10-mer peptides, the fragment can be 11 amino acidresidues in length. Beginning with a fragment comprising amino acid1-11, and progressing to the C-terminal residue, 122 different peptidesare possible for the corresponding 132 amino acid region in nsp2.Similarly, the fragment can be 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore residues in length, up to 131 residues in length, and such peptidesare readily identifiable by beginning at residue 1 and “walking” downthe amino acid sequence of the corresponding peptide. There are 7626such fragments in a peptide of 132 amino acid residues (including thefullength peptide). Such a strategy can be applied to the correspondingnsp2 sequence from any strain of PRRS virus, making it possible to havea diagnostic kit specific for that and possibly other related strains ofPRRS virus.

Example XIII Infection of Pigs with Recombinant Viruses

To establish the level of attenuation achieved by the deletion of thensp2 (628-759) peptide, and to evaluate the ability of the deletion toserve as a serological marker, young pigs are infected with theundeleted parental virus P129-Mlu/SgrA, P129-Mlu-GFP-SgrA orP129-Mlu-HA-SgrA. Three groups of 10 conventional weaned pigs, 3 weeksof age, are housed separately. A mock-infected group (6 pigs) isincluded in the study, and naïve pigs (contact controls, 2 per vaccinegroup) are commingled with infected pigs in order to evaluate theability of the viruses to spread. After a week of acclimation, at about4 weeks of age, pigs are infected with the three viruses, or mockinfected, by delivering 2 ml of a diluted virus stock (at 1×10⁴TCID₅₀/ml) intramuscularly and 1 ml intranasally to each nostril (totaldose is 4×10⁴ TCID₅₀/ml). Pigs are monitored daily for clinical signs ofdisease from day −3 to day 10 (general condition, depression, loss ofappetite, sneezing, coughing, and respiratory distress), and rectaltemperatures are taken on day −1, 3, 7, and 10. On day 10, half of thepigs in each group (selected by prior allotment) are necropsied in orderto examine lung lesions and to sample tissues for virus isolation andhistopathology. The remaining pigs are necropsied on day 28. All pigsare weighed on arrival, on day −1 or 0, and at necropsy (day 10 or 28).

Blood (10 ml) is collected from all pigs on days −1 or 0, 3, 7, 10, 14,21, and 28, into serum separator tubes. Sera are frozen at −80 C untilused for the determination of anti-PRRSV antibodies (by IDEXX HerdChekELISA), neutralizing antibodies (by serum neutralization assay), andvirus isolation (titration of virus infectivity by TCID₅₀ assay). Inaddition, sera will be assayed for antibodies against the nsp2 (628-759)peptide using the ELISA described in Example XII above. Also, theinduction of serum antibodies to GFP or to the HA tag can be determinedusing commercial kits or reagents.

The viruses with nsp2 (628-759) deleted are attenuated (less virulent)relative to the parental virus P129-Mlu/SgrA, as evidenced by acombination of one or more of the following characteristics in infectedpigs: (1) reduced clinical signs, (2) reduced fever, (3) increasedweight gain, (4) reduced number or severity of lung lesions, (5)increased neutralizing antibody levels, (6) decreased level or durationof viremia in serum, lung, and/or tonsil tissue, or (7) reduced spreadto naïve contact control pigs. Attenuated viruses with thesecharacteristics are useful as live vaccines.

All infected pigs test positive for anti-PRRSV antibodies using acommercial ELISA (IDEXX HerdChek), with seroconversion occurring nolater than 21 days post-infection. Only those pigs infected withundeleted parental virus P129-Mlu/SgrA are expected to have antibodiesspecific to the nsp2 (628-759) peptide, as measured using the ELISAdescribed in Example XII. Such a vaccine virus is useful fordifferentiating infected from vaccinated animals (DIVA), otherwise knownas a negatively marked vaccine.

Sera from pigs infected with P129-Mlu-GFP-SgrA or P129-Mlu-HA-SgrA areassayed for antibodies to GFP and the HA tag, respectively, by detectingthese antibodies using commercially available reagents and kits. Suchpositive serological markers or “compliance markers”, when incorporatedinto a vaccine, are useful for determining which pigs in a herd havebeen successfully immunized with the vaccine. In addition, the waning ofantibodies against the marker can be used as surrogate for the waning ofantibodies against the PRRS virus, giving an indication of when torevaccinate.

In a separate pig study, the ability of an nsp2 (628-759)-deleted PRRSvirus to function as a vaccine is tested directly. The P129-PK-dnsp2virus (Example XI) is used to immunize 4 week old pigs by delivering 2ml of virus at 2×10⁴ TCID₅₀/ml intramuscularly. After 28 days, thesepigs are challenged by infection with a virulent PRRS isolate (NADC20).The severity of the disease induced by the NADC20 virus is compared todisease induced in non-vaccinated (naïve) pigs. A useful vaccine willresult in at least partial reduction of disease caused by NADC20,including clinical signs, fever, reduced weight gain, lung lesions,persistent viremia, and spread to contact control pigs.

DEPOSIT OF BIOLOGICAL MATERIALS

The following biological material was deposited with the American TypeCulture Collection (ATCC) at 10801 University Blvd., Manassas, Va.,20110-2209, USA, on Nov. 19, 1998 and were assigned the followingaccession numbers:

Plasmid Accession No. pT7P129A 203488 pCMV-S-P129 203489

All patents, patent applications, and publications cited above areincorporated herein by reference in their entirety.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

1. An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that when it infects a porcine animal it is unable to produce PRRS in the animal yet able to elicit an effective immunoprotective response against a PRRS virus in the porcine animal, wherein said DNA sequence is SEQ ID NO:1 or a sequence homologous thereto, except that it lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus.
 2. The isolated polynucleotide molecule of claim 1, wherein said molecule lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus in ORF 1a or ORF 1b within said DNA sequence.
 3. The isolated polynucleotide molecule of claim 1, wherein said molecule lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
 4. The isolated polynucleotide molecule of claim 1, wherein said molecule lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
 5. The isolated polynucleotide molecule of claim 1, wherein said molecule lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus between amino acids from 616 to 752 in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
 6. The isolated polynucleotide molecule of claim 1, wherein said molecule lacks the DNA sequence encoding amino acids 628 and 759 in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
 7. A vaccine for protecting a porcine animal from infection by a PRRS virus, wherein said vaccine comprises: a) A genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by the isolated polynucleotide molecule according to claim 1; b) An infectious RNA molecule encoded by the isolated polynucleotide molecule according to claim 1; c) The isolated polynucleotide molecule according to claim 1 in the form of a plasmid, or d) A viral vector comprising the isolated polynucleotide molecule according to claim 1; and a carrier acceptable for veterinary use.
 8. The isolated polynucleotide molecule of claim 1, wherein said isolated polynucleotide molecule further comprises at least one nucleotide sequence that encodes a detectable heterologous antigenic epitope.
 9. The isolated polynucleotide molecule of claim 8, wherein translation of the encoded infectious RNA molecule results in formation of at least one protein consisting of a fusion between a PRRS virus protein and said heterologous antigenic epitope.
 10. The isolated polynucleotide molecule of claim 8, wherein said nucleotide sequence that encodes a heterologous antigenic epitope is inserted in ORF 1a or ORF 1b within said DNA sequence.
 11. The isolated polynucleotide molecule of claim 8, wherein said nucleotide sequence that encodes a heterologous antigenic epitope is inserted in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
 12. The isolated polynucleotide molecule of claim 8, wherein said nucleotide sequence that encodes a heterologous antigenic epitope is inserted in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
 13. The isolated polynucleotide molecule of claim 8, wherein said nucleotide sequence that encodes a heterologous antigenic epitope is inserted between amino acids from 628 to 759 in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
 14. The isolated polynucleotide molecule of claim 8, wherein said nucleotide sequence that encodes a heterologous antigenic epitope is inserted between amino acids from 616 to 752 in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
 15. A vaccine for protecting a porcine animal from infection by a PRRS virus, wherein said vaccine comprises: a) A genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by the isolated polynucleotide molecule according to claim 8; b) An infectious RNA molecule encoded by the isolated polynucleotide molecule according to claim 8; c) The isolated polynucleotide molecule according to claim 8 in the form of a plasmid, or d) A viral vector comprising the isolated polynucleotide molecule according to claim 8; and a carrier acceptable for veterinary use.
 16. A diagnostic kit comprising reagents which are useful for differentiating between porcine animals naturally infected with a field strain of a PRRS virus and porcine animals vaccinated with the vaccine of claim
 15. 17. The diagnostic kit of claim 15 having as one of its components a peptide comprising the amino acid sequences selected from SEQ ID NO. 64 to
 115. 18. The diagnostic kit of claim 16, wherein one of its components is a peptide comprising the amino acid sequence of SEQ ID NO. 64, or a peptide fragment thereof 10 amino acid residues in length or longer.
 19. The diagnostic kit of claim 17, wherein said peptide is optionally a fusion protein. 